Immunotherapy against erbb-3 receptor

ABSTRACT

The present invention describes methods and pharmaceutical compositions for the treatment of cancer in mammals, more particularly in human subjects. More specifically, the invention concerns anti-tumor vaccines based upon plasmid DNA and/or genetic vectors carrying a codon-usage optimized sequence and coding for a mutant form of the ErbB-3 receptor. Furthermore, the invention refers to monoclonal antibodies directed against the ErbB-3 receptor, obtained using these methods and capable to block its activity in cancer cells.

The present invention describes methods and pharmaceutical compositionsfor the treatment of cancer in mammals, more particularly in humansubjects. More specifically, the invention concerns anti-tumor vaccinesbased upon plasmid DNA and/or genetic vectors carrying a codon-usageoptimized sequence and coding for a mutant form of the ErbB-3 receptor.Furthermore, the invention refers to monoclonal antibodies directedagainst the ErbB-3 receptor, obtained using these methods and capable toblock its activity in cancer cells.

BACKGROUND OF THE INVENTION

Research activities in oncology are increasingly directed to identifynovel treatments with enhanced specificity against tumors and directedagainst precise molecular targets (targeted therapy). Indeed veryfrequently in tumors it is observed that uncontrolled cell growth is dueto altered processes of signal transduction from the cell surface thatinduce cell proliferation, inhibit apoptosis and which frequentlyinvolve overexpression and activation of specific surface receptors. TheEGFR family (otherwise called ErbB or HER) is constituted by fourtransmembrane proteins (EGFR/HER1, HER2, HER3 and HER4) which playmultiple roles both in normal cells and in the development andmaintenance of tumors [1]. Three aberrant mechanisms contribute to thetumorigenic activity of ErbB receptors: receptor overexpression, oftenlinked to gene amplification; constitutive activation due to specificmutation, ligand overexpression. Given the key role of these receptorsin oncogenic signalling, new drugs have been developed, both monoclonalantibodies and small molecules, having as targets in particular EGFR andHER2, and which are widely employed in clinical protocols for thetherapy of a variety of tumors, mainly lung, colon and breast. Amongthese, antibodies like cetuximab, panitumumab (against EGFR),trastuzumab and pertuzumab (against HER2) and tyrosine-kinase inhibitors(TKIs) gefitinib, erlotinib (against EGFR) and lapatinib (againstEGFR/HER2) [1]. However, in spite of these progresses, the clinicalefficacy of these agents is lower than expected and often is accompaniedby the emergence of resistance. For example objective responses observedwith trastuzumab (Herceptin™) in patients with HER2 positive mammarytumors are low (in general 15%) and short lived [2]. Furthermore,several prototype tumor vaccines against HER2 based mainly upon the useof peptides and proteins have been developed over the last years andtested in Phase I and II clinical trials, without significant results[3,4]. These vaccines had been designed to induce mainly a cell-mediatedimmune response, with a principal involvement of cytotoxic CD8+ cells(CTLs). The generation of CTL correlated with the prevention anderadication of HER2+ tumor cells in preclinical models, but was unableto control the diffusion of metastasis in human patients. Subsequently,some studies have demonstrated that tumor cells treated simultaneouslywith trastuzumab and with CTLs derived from patients vaccinated withpeptides were lysed more efficiently [5], thus suggesting that anantitumor vaccine against HER2 capable of inducing simultaneously bothan antibody response and a cytotoxic response should be able to achievea significant enhancement of therapeutic efficacy. More recently,genetic vaccines against HER2 based upon plasmid DNA electroporationinto muscle tissue and the use of recombinant Adenoviral vectors havebeen shown to be able to achieve these goals and have provided promisingresults [6-8], being able to induce at the same time the development ofinnate immunity, cell-mediated immunity and, most importantly, hightiter antibody responses against the receptor.

A distinctive feature of the members of the ErbB receptor family is theinterdependence and complementarity of their functions. While HER2 is aknown oncogene and its overexpression, mainly linked to geneamplification in approximately 25% of breast tumors, has been causallycorrelated to tumor development [9], for HER3 no mutations have beenfound to be directly involved in the process of carcinogenesis. However,loss of HER3 expression abolishes the transforming ability of HER2.Hence, HER3 can be considered as an obligate partner of HER2-mediatedtransformation [1,10]. Furthermore HER3 seems to play a key role in thedevelopment of resistance to current EGFR and HER2 inhibitors, mostlikely as a consequence of its overexpression and increased plasmamembrane localization: its role appears to be linked to the formation ofheterodimers with EGFR and with HER2 and its ability to betransphosphorylated in six tyrosine residues that serve as binding sitesfor molecules involved in the downstream signalling, such as p85, theregulatory subunit of PI3K [11]. Also cMet amplification has recentlybeen described in cells resistant to TKIs, and under this circumstance,the mechanism of resistance seems to be mediated by HER3transphosphorylation by overexpressed cMet [12]. The PI3K/Akt pathway iscritical for the viability and maintenance of cancer stem cells inbreast [13], prostate [14], lung [15], colon [16], brain [17] cancers aswell as in leukemias [18]. Given the central role of PI3K in thesignalling in cancer stem cells and the inability of HER2 to activatethe PI3K axis in the absence of HER3, it can be hypothesized that HER3plays a fundamental role in cancer stem/progenitor cells. Hence, itsinhibition may be a powerful strategy to eradicate these cells andimprove efficacy of current therapies.

HER3 consists of an extracellular domain which binds to the ligand(ECD), a dimerization domain within the ECD, a transmembrane domain (TM)and a C-terminal domain (ICD), which is phosphorylated. Neuregulin (NRG)or other ligands bind the ECD and trigger signal transduction promotingreceptor dimerization with other RTKs and ICD transphosphorylation.Because HER3 does not possess tyrosine kinase activity, its function canonly be inhibited by specific monoclonal antibodies. In literature,there are already evidences that antibodies directed against HER3 candisplay antitumor activity. Schoeberl and coworkers [19,20] haverecently shown in primary tumors and in cell lines that express membersof the ErbB family and relevant ligands, hence with autocrine loops,that only anti-HER3 antibodies but not antibodies against EGFR(cetuximab), or HER2 (trastuzumab and pertuzumab), are capable to fullyinhibit receptor activation induced by all ligands of this receptorfamily, whereas cetuximab and trastuzumab are able to neutralize only asubset of them.

SUMMARY OF THE INVENTION

In a first aspect the present invention is directed at nucleic acidsencoding a variant of HER3, vectors comprising such nucleic acids andmethods and tools to block the oncogenic activity of HER3 and ofreceptors of the ErbB family by using such nucleic acids and vectors.Thus, in a first embodiment, the invention is directed at a new methodto induce an immune response against HER3. In particular, the methoduses a genetic vaccination which utilizes a vector, for example plasmidDNA, carrying the optimized cDNA for human HER3, expressing the proteinwith a H584F single amino acid mutation. Such a vector is preferablyinjected intramuscularly, and following that administration it ispreferred that an electric field is applied in order to increase theexpression level of the antigen, and to induce in the host organism anantibody and/or cell-mediated immune response against HER3. Such avaccine, here identified, can be utilized as monotherapy or incombination with chemotherapy (e.g. cisplatin, irinotecan, oxaliplatin),monoclonal antibodies (e.g. Trastuzumab, Pertuzumab, Cetuximab,anti-HER3 antibodies), TKI agents (e.g. gefitinib, erlotinib,lapatinib), immunomodulators (e.g. TLR agonists, MF-59, ipilimumab orother monoclonal antibodies directed against CTLA4) and other antitumorvaccines (e.g. Provenge™, GVAX™)

In a further aspect, the invention is directed to antagonisticmonoclonal antibodies generated by the vaccine above which bind theextracellular domain of HER3 and the receptor-dependent signaltransduction events, such as pHER3, pAKT and cell proliferation. Thenucleotide and amino acid sequence and the process for the generation ofthese antibodies are described. Expression vectors and host cells whichcontain them for the production of the antibodies of the invention arealso described in detail. Also these biological agents can findapplication as monotherapy or in combination with other anticancertherapeutics, preferably chemotherapy, other antibodies, TKI agents,immunomodulators and antitumor vaccines. A significant advantage of theinvention is based upon the antibody specificity, with known potency.The affinity of the antibodies obtained with this new technology issurprisingly higher than other known antibodies. It is expected thatthese antibodies can be efficacious in the treatment of patients whohave developed resistance to tyrosine-kinase inhibitors, such as TKIs,anti-EGFR antibodies, anti-HER2 antibodies and agents against cMet.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs.

Preferably, the terms used herein are defined as described in “Amultilingual glossary of biotechnological terms: (IUPACRecommendations)”, Leuenberger, H. G. W, Nagel, B. and Kölbl, H. eds.(1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

To practice the present invention, unless otherwise indicated,conventional methods of chemistry, biochemistry, cell biology, andrecombinant DNA techniques are employed which are explained in theliterature in the field (cf., e.g., Molecular Cloning: A LaboratoryManual, 2^(nd) Edition, J. Sambrook et al. eds., Cold Spring HarborLaboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps. Asused in this specification and the appended claims, the singular forms“a”, “an”, and “the” include plural referents, unless the contentclearly dictates otherwise.

The term “about” when used in connection with a numerical value is meantto encompass numerical values within a range having a lower limit thatis 5% smaller than the indicated numerical value and having an upperlimit that is 5% larger than the indicated numerical value.

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including all patents, patentapplications, scientific publications, manufacturer's specifications,instructions, etc.), whether supra or infra, are hereby incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

An “amino acid substitution” comprises the replacement of one or moreamino acids in a protein with another. Substitutions may preserve,diminish or eliminate the protein function. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent or degree of similarity may be adjusted upwards to correct forthe conservative nature of the substitution. Means for making thisadjustment are well known to those of skill in the art. See, e.g.,Pearson [33]. Examples of groups of amino acids that have side chainswith similar chemical properties include

-   -   1) aliphatic side chains: glycine, alanine, valine, leucine and        isoleucine;    -   2) aliphatic-hydroxyl side chains: serine and threonine;    -   3) amide-containing side chains: asparagine and glutamine;    -   4) aromatic side chains: phenylalanine, tyrosine, and        tryptophan;    -   5) basic side chains: lysine, arginine, and histidine;    -   6) acidic side chains: aspartate and glutamate, and    -   7) sulfur-containing side chains: cysteine and methionine.

Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamate-aspartate, and asparagine-glutamine.Alternatively, a conservative replacement is any change having apositive value in the PAM250 log-likelihood matrix disclosed in Gonnetet al. [34]. A “moderately conservative” replacement is any changehaving a nonnegative value in the PAM250 log-likelihood matrix. Giventhe known genetic code, and recombinant and synthetic DNA techniques,the skilled scientist can readily construct DNAs encoding conservativeamino acid variants. A “non-conservative substitutions” or“non-conservative amino acid exchanges” are defined as exchanges of anamino acid by another amino acid listed in a different group of theseven standard amino acid groups 1) to 7) shown above.

The term “codon”, as used herein, refers to a sequence of threenucleotides that encode a specific amino acid within the genetic code.

As used herein, the term “expression vector”, refers to an expressionconstruct, which comprises elements for promoting transcription of adesired coding sequence. Preferred examples of expression vectors areselected from the group consisting of a bacterial plasmid, anadenovirus, a poxvirus, a vaccinia virus, a fowlpox virus, a herpesvirus, an adeno-associated virus (AAV), an alphavirus, a lentivirus, alambda phage, a lymphocytic choriomeningitis virus and a Listeria sp,Salmonella sp., used to introduce a specific gene into a target cell.Expression vectors comprise plasmids as well as viral vectors andgenerally contain a desired coding sequence and appropriate DNAsequences necessary for the expression of the operably linked codingsequence in a particular host organism (e.g., bacteria, yeast, plant,insect, or mammal) or in in vitro expression systems. Cloning vectorsare generally used to engineer and amplify a certain desired DNAfragment and may lack functional sequences needed for expression of thedesired DNA fragments. Once the expression vector is inside the cell,the protein is encoded by the gene. In this application a plasmid vectorhas been utilized which carries a modified cDNA.

The term “promoter”, refers to a DNA region that facilitates thetranscription of a particular gene. Promoters are located upstream ofthe regulated gene (towards the 5′ region of the sense strand) andrepresent critical elements that can work in concert with otherregulatory transcriptional elements (e.g. enhancers, silencers, boundaryelements, insulators) to direct the level of transcription of a givengene. The term “regulatory transcriptional element” refers to e.g. corepromoters, proximal promoters, distal enhancers, silencers,insulators/boundary elements (see, e.g., Maston et al. [35]).

The term “neoplasm” refers to an abnormal mass of tissue as a result ofneoplasia which is the abnormal proliferation of cells. If the growth ofneoplastic cells exceeds and is not coordinated with that of the normaltissues around it, neoplasm can causes a tumor and/cancer.

The term “genetic vaccine” or “genetic vaccination”, as used herein,refers to non-living vaccines that trigger a full immune response. Itcomprises the direct injection of genetic material into a living hostresulting in a small amount of its cells to express the introduced geneproducts and further resulting in a specific immune activation of thehost against the gene delivered antigen (see, e.g., Koprowski et al.[36]).

The term “electroporation” refers to a significant increase in theelectrical conductivity and permeability of the cell plasma membranecaused by an externally applied electrical field. In this applicationthe method is used in a preferred embodiment to introduce DNA into acell according to [21]. Electroporation can be applied in vitro or invivo.

The term “antibody” typically refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen-binding portion thereof. The term“antibody” also includes all recombinant forms of antibodies. Each heavychain is comprised of a heavy chain variable region (abbreviated hereinas VH or V_(H)) and a heavy chain constant region. Each light chain iscomprised of a light chain variable region (abbreviated herein as VL orV_(L)) and a light chain constant region. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR). Each VH and VL is composed ofthree CDRs, arranged from amino-terminus to carboxy-terminus in thefollowing order: CDR1, CDR2 and CDR3.

The term “antigen-binding fragment” of an antibody (or simply “bindingportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen. Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of a full-length antibody, e.g. via isolatedcomplementarity determining regions (CDRs), and combinations of two ormore isolated CDRs which may optionally be joined by a synthetic linker.Other examples include small antibody mimetics comprising two or moreCDR regions that are fused to each other, preferably by cognateframework regions. Such a small antibody mimetic comprising V_(H) CDR1and V_(L) CDR3 linked by the cognate V_(H) FR2 has been described by Qiuet al. [37].

Antibodies and antigen-binding fragments thereof usable in the inventionmay be from any animal origin including mammals. Preferably, theantibodies or fragments are from human, chimpanzee, rodent (e.g. mouse,rat, guinea pig, or rabbit), chicken, turkey, pig, sheep, goat, camel,cow, horse, donkey, cat, or dog origin. It is particularly preferredthat the antibodies are of human or murine origin. Antibodies of theinvention may also include chimeric molecules in which an antibodyconstant region derived from one species, preferably human, is combinedwith the antigen binding site derived from another species, e.g. mouse.Moreover antibodies of the invention include humanized molecules inwhich the antigen binding sites of an antibody derived from a non-humanspecies (e.g. from mouse) are combined with constant and frameworkregions of human origin.

The term “chimeric antibody” refers to those antibodies wherein oneportion of each of the amino acid sequences of heavy and light chains ishomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular class, while theremaining segment of the chain is homologous to corresponding sequencesin another species or class.

The term “humanized antibody” refers to a molecule having an antigenbinding site that is substantially derived from an immunoglobulin from anon-human species, wherein the remaining immunoglobulin structure of themolecule is based upon the structure and/or sequence of a humanimmunoglobulin. The antigen binding site may either comprise completevariable domains fused onto constant domains or only the complementaritydetermining regions (CDR) grafted onto appropriate framework regions inthe variable domains. Antigen-binding sites may be wild-type or modifiedby one or more amino acid substitutions, e.g. modified to resemble humanimmunoglobulins more closely. Some forms of humanized antibodiespreserve all CDR sequences (for example a humanized mouse antibody whichcontains all six CDRs from the mouse antibody). Other forms have one ormore CDRs which are altered with respect to the original antibody.

Different methods for humanizing antibodies are known to the skilledperson, as reviewed by Almagro et al. [38], the content of which isherein incorporated by reference in its entirety. The review article byAlmagro et al. [38] is briefly summarized in the following. Almagro etal. [38] distinguish between rational approaches and empiricalapproaches. Rational approaches are characterized by generating fewvariants of the engineered antibody and assessing their binding or anyother property of interest. If the designed variants do not produce theexpected results, a new cycle of design and binding assessment isinitiated. Rational approaches include CDR grafting, Resurfacing,Superhumanization, and Human String Content Optimization. In contrast,empirical approaches are based on the generation of large libraries ofhumanized variants and selection of the best clones using enrichmenttechnologies or high-throughput screening. Accordingly, empiricalapproaches are dependent on a reliable selection and/or screening systemthat is able to search through a vast space of antibody variants. Invitro display technologies, such as phage display and ribosome displayfulfill these requirements and are well-known to the skilled person.Empirical approaches include FR libraries, Guided selection,Framework-shuffling, and Humaneering.

CDR Grafting

A CDR grafting protocol typically comprises three decision-makingpoints: (1) definition of regions determining the specificity of thedonor antibody, i.e. the target for grafting, (2) identification of asource of human sequences to be utilized as FR donors, and (3) selectionof residues outside of the region defining the specificity, i.e.determining amino acid positions that are targets for back mutation torestore or improve the affinity of the humanized antibody.

(1) Regions Determining the Antibody Specificity

The experimental structure of the non-human antibody in complex with theantigen provides a detailed map of residues in contact with the antigenand therefore those responsible for determining its specificity. Thestructural information can be complemented with alanine scanningmutagenesis and/or combinatorial mutagenesis to identify the residuescontributing most to the binding energy or to the functional paratope.Since the functional paratope is a subset of the residues in contact,grafting only the functional paratope would reduce the number ofnon-human residues in the humanized product. However, only in rare casesare the experimental structure of the antigen-antibody complex and/orthe functional paratope available at the beginning of a humanizationprotocol. In absence of a precise definition of residues responsible fora given antibody specificity, CDRs are often employed as regionsdefining the specificity. It is also possible to use a combination ofCDR and HV loop as targets for grafting. To reduce the number ofresidues to be grafted on the human FRs, SDR grafting has beendescribed, i.e. the grafting of specificity-determining residues (SDRs).

(2) Source of Human FRs

Frame work (FR) regions are more conserved regions within light chainsand heavy chain of an antibody. The second step in a typical CDRgrafting protocol is to identify human FR donors. Initial works utilizedFRs of human antibodies of known structure, regardless of their homologyto the non-human antibody. This approach is known as “Fixed FR method”.Later works used human sequences having the highest homology to thenon-human antibody. This approach has been termed “Best Fit”. While“best fit” strategies tend to result in antibodies with higher affinity,other parameters such as low immunogenicity and production yields haveto be taken into account, too, when choosing an FR for humanization.Thus, combinations of “best fit” and “fixed FR” are also possible. Forexample, the V_(L) part can be humanized according to the fixed FRmethod and the V_(H) part can be humanized according to the best fitmethod, or vice versa.

Two sources of human sequences have been utilized: mature and germlinesequences. Mature sequences, which are products of immune responses,carry somatic mutations generated by random processes and are not underthe species selection, resulting in potential immunogenic residues.Thus, to avoid immunogenic residues, human germline genes haveincreasingly been utilized as source of FR donors. Nucleotide sequencesof human germline FRs are disclosed e.g. in Appendices A and B of thearticle by Dall'Acqua et al. [39]. Furthermore, germline gene basedantibodies tend to be more flexible as compared to mature antibodies.This higher flexibility is thought to better accommodate diverse CDRswith fewer or no back mutations into the FR to restore the affintiy ofthe humanized antibody.

(3) Back Mutations to Restore or Enhance Affinity

Commonly, affinity decreases after CDR grafting as a consequence ofincompatibilities between non-human CDRs and human FRs. Therefore, thethird step in a typical CDR grafting protocol is to define mutationsthat would restore or prevent affinity losses. Back mutations have to becarefully designed based on the structure or a model of the humanizedantibody and tested experimentally. A web site for automated antibodymodeling called WAM can be found at the URL http://antibody.bath.ac.uk.Software for protein structure modeling can be downloaded at the siteshttp://salilab.org/modeller/modeller.html (Modeller) andhttp://spdbv.vital-it.ch (Swiss PdbViewer).

Resurfacing

Resurfacing is similar to CDR grafting and shares the first twodecision-making points. In contrast to CDR grafting, resurfacing retainsthe non-exposed residues of the non-human antibody. Only surfaceresidues in the non-human antibody are changed to human residues.

Superhumanization

While CDR grafting relies on the FR comparison between the non-human andthe humans sequences, superhumanization is based on a CDR comparison sothat FR homology is irrelevant. The approach includes a comparison ofthe non-human sequence with the functional human germline generepertoire. Those genes encoding the same or closely related canonicalstructures to the murine sequences are then selected. Next, within thegenes sharing the canonical structures with the non-human antibody,those with highest homology within the CDRs are chosen as FR donors.Finally, the non-human CDRs are grafted onto these FRs [31]. This methodis preferred within this application.

Human String Content Optimization

This approach is based on a metric of antibody “humanness”, termed HumanString Content (HSC). In short, this approach compares the mousesequence with the repertoire of human germline genes. Differences arescored as HSC. The target sequence is the humanized by maximizing itsHSC rather than using a global identity measure to generate multiplediverse humanized variants.

Framework Libraries (Abbreviated: FR Libraries)

In the FR library approach, a collection of residue variants areintroduced at specific positions in the FR followed by panning of thelibrary to select the FR that best supports the grafted CDR. Thus, thisapproach resembles CDR grafting but instead of creating a few backmutations in the FR, a combinatorial library of typically more than 100mutational variants is constructed.

Guided Selection

This approach includes combining the V_(H) or V_(L) domain of a givennon-human antibody specific for a particular antigen with a human V_(H)and V_(L) library. Subsequently, specific human V domains are selectedagainst the antigen of interest. For example, a non-human antibody canbe humanized by first combining the non-human V_(H) with a library ofhuman light chains. The library is then selected against the targetantigen by phage display and the selected V_(L) is cloned into a libraryof human V_(H) chains and selected against the target antigen. It isalso possible to start with combining the non-human V_(L) with a libraryof human heavy chains. The library is then selected against the targetantigen by phage display and the selected V_(H) is cloned into a libraryof human V_(L) chains and selected against the target antigen. As aresult, a fully human antibody with similar affinity as the non-humanantibody can be isolated. To avoid the occurrence of an epitope drift,it is possible to implement a so-called inhibition ELISA, which allowsfor the selection of clones recognizing the same epitope as the parentantibody. Alternatively, CDR retention can be applied to avoid anepitope drift. In CDR retention, one or more non-human CDRs areretained, preferably the heavy chain CDR3, since this CDR is at thecenter of the antigen binding site.

Framework Shuffling (Abbreviated: FR Shuffling)

In the FR shuffling approach, whole FRs are combined with the non-humanCDRs. Using FR shuffling, Dall'Acqua and co-workers humanized a murineantibody. All six CDRs of the murine antibody were cloned into a librarycontaining all human germline gene FRs (Dall'Acqua et al. [39]). Thelibraries were screened for binding in a two-step selection process,first humanizing V_(L), followed by V_(H). In a later study, a one-stepFR shuffling process was successfully used (Damschroder et al. [40]).Oligonucleotide sequences encoding all known human germline light chain(κ) frameworks are disclosed in Dall'Acqua et al. [39], as Appendix A.Oligonucleotide sequences encoding all known human germline heavy chainframeworks are disclosed in Dall'Acqua et al. [39].

Humaneering

Humaneering allows for isolation of antibodies that are 91-96%homologous to human germline gene antibodies. The method is based onexperimental identification of essential minimum specificitydeterminants (MSDs) and on sequential replacement of non-human fragmentsinto libraries of human FRs and assessment of binding. It begins withregions of the CDR3 of non-human V_(H) and V_(L) chains andprogressively replaces other regions of the non-human antibody into thehuman FRs, including the CDR1 and CDR2 of both V_(H) and V_(L).

The methods for humanizing antibodies explained above are preferred whengenerating humanized antibodies that specifically bind to conformationalepitopes. Nevertheless, the present invention is not limited to theabove-mentioned methods for humanizing antibodies.

Some of the aforementioned humanization methods can be performed withoutinformation about the FR sequences in the donor antibody, namely the“Fixed FR Method” (a variant of CDR-grafting), Superhumanization,Framework-shuffling, and Humaneering. Variations of the “fixed FRmethod” were successfully carried out by Qin et al. [41] and Chang etal. [42]. In particular, Qin et al. [41] constructed an antibodyfragment comprising a human heavy chain variable region in which thethree CDR regions were replaced by antigenic peptides, which werederived from the CDR sequences of a murine antibody. Chang et al. [42]continued these experiments and constructed an scFv fragment, in whichall CDRs from the V_(H) part and CDR3 from the V_(L) part were replacedby antigenic peptides, which were derived from the CDR sequences of amurine antibody.

As used herein, “human antibodies” include antibodies having variableand constant regions derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo). Human antibodies of the invention includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulin and that do not expressendogenous immunoglobulins, as described for example in U.S. Pat. No.5,939,598 by Kucherlapati & Jakobovits.

The term “monoclonal antibody” as used herein refers to a preparation ofantibody molecules of single molecular composition. A monoclonalantibody displays a single binding specificity and affinity for aparticular epitope.

The term “recombinant antibody”, as used herein, includes all antibodiesthat are prepared, expressed, created or isolated by recombinant means,such as (a) antibodies isolated from an animal (e.g., a mouse) that istransgenic or transchromosomal with respect to the immunoglobulin genesor a hybridoma prepared therefrom, (b) antibodies isolated from a hostcell transformed to express the antibody, e.g. from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial antibody library,and (d) antibodies prepared, expressed, created or isolated by any othermeans that involve splicing of immunoglobulin gene sequences to otherDNA sequences.

The term “identity” or “identical,” when referring to a nucleic acid orfragment thereof, indicates that, when optimally aligned withappropriate nucleotide insertions or deletions with another nucleic acid(or its complementary strand), there is nucleotide sequence identity inat least about 80%, and more preferably at least about 85%, 90%, 95%,96%, 97%, 98% or 99% of the nucleotide bases, as measured by anywell-known algorithm of sequence identity, such as FASTA, BLAST or GAP,as discussed below.

As applied to polypeptides, the term “identity of XX %” or “identical”means that two peptide sequences, when optimally aligned, such as by theprograms GAP or BESTFIT using default gap weights, share the indicatedpercentage of identical amino acids. Preferably, the amino acids shareat least 80%, more preferably at least 85%, at least 90% sequenceidentity, even more preferably at least 95%, 98% or 99% sequenceidentity. Preferably, residue positions which are not identical differby conservative amino acid substitutions.

Sequence similarity for polypeptides is typically measured usingsequence analysis software. Protein analysis software matches similarsequences using measures of similarity assigned to varioussubstitutions, deletions and other modifications, including conservativeamino acid substitutions. For instance, GCG software contains programssuch as GAP and BESTFIT which can be used with default parameters todetermine sequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild type protein and a mutant thereof, see e.g.GCG Version 6.1. Polypeptide sequences also can be compared using FASTAwith default or recommended parameters; a program in GCG Version 6.1.FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequenceidentity of the regions of the best overlap between the query and searchsequences (see Pearson [43]). Another preferred algorithm when comparinga sequence of the invention to a database containing a large number ofsequences from different organisms is the computer program BLAST,especially BLASTP or TBLASTN, using default parameters, see e.g.Altschul et al. [44] which is herein incorporated by reference.

When percentages of sequence identity are referred to in the presentapplication, these percentages are calculated in relation to the fulllength of the longer sequence, if not specifically indicated otherwise.This calculation in relation to the full length of the longer sequenceapplies both to nucleic acid sequences and to polypeptide sequences.

The identity in the sequences may be assessed by aligning thepolypeptide sequences. Such alignment tools are well known to the personskilled in the art and can be, for example, obtained on the World WideWeb, e.g., ClustalW (www.ebi.ac.uk/clustalw) or Align(http://www.ebi.ac.uk/_emboss/align/_index.html) using standardsettings, preferably for Align EMBOSS::needle, Matrix: Blosum62, GapOpen 10.0, Gap Extend 0.5. The “best sequence alignment” between twopolypeptides is defined as the alignment that produces the largestnumber of aligned identical residues.

In the context of the present invention it is stated that one or moreresidues in a polypeptide “occupy an analogous position” with respect toone or more residues in a reference polypeptide. It is well known in theart, that analogous positions between a reference polypeptide and one ormore further polypeptides can be determined by aligning the polypeptidesequences based on amino acid sequence or structural similarities. Suchalignment tools are well known to the person skilled in the art and canbe, for example, obtained on the World Wide Web, e.g., ClustalW(www.ebi.ac.uk/clustalw) or Align(http://www.ebi.ac.uk/emboss/align/index.html) using standard settings,preferably for Align EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0,Gap Extend 0.5. Those skilled in the art understand that it may benecessary to introduce gaps in either sequence to produce a satisfactoryalignment. Once the alignment is completed the relevant residue orresidues of the reference polypeptide are identified and the residue orresidues of the one or more polypeptides that are aligned withthis/these residues can be determined.

As used herein, “treat”, “treating” or “treatment” of a disease ordisorder means accomplishing one or more of the following: (a) reducingthe severity and/or duration of the disorder; (b) limiting or preventingdevelopment of symptoms characteristic of the disorder(s) being treated;(c) inhibiting worsening of symptoms characteristic of the disorder(s)being treated; (d) limiting or preventing recurrence of the disorder(s)in patients that have previously had the disorder(s); and (e) limitingor preventing recurrence of symptoms in patients that were previouslysymptomatic for the disorder(s).

As used herein, “prevent”, “preventing”, “prevention”, or “prophylaxis”of a disease or disorder means preventing that a disorder occurs insubject.

As used herein, a first compound (e.g. an antibody) is considered to“bind” to a second compound (e.g. an antigen, such as a target protein),if it has a dissociation constant K_(d) to said second compound of 1 mMor less, preferably 100 μM or less, preferably 50 μM or less, preferably30 μM or less, preferably 20 μM or less, preferably 10 μM or less,preferably 5 μM or less, more preferably 1 μM or less, more preferably900 nM or less, more preferably 800 nM or less, more preferably 700 nMor less, more preferably 600 nM or less, more preferably 500 nM or less,more preferably 400 nM or less, more preferably 300 nM or less, morepreferably 200 nM or less, even more preferably 100 nM or less, evenmore preferably 90 nM or less, even more preferably 80 nM or less, evenmore preferably 70 nM or less, even more preferably 60 nM or less, evenmore preferably 50 nM or less, even more preferably 40 nM or less, evenmore preferably 30 nM or less, even more preferably 20 nM or less, andeven more preferably 10 nM or less, even more preferably 8 nM or less,most preferred 8 nM or less.

The term “binding” according to the invention preferably relates to aspecific binding. “Specific binding” means that a binding moiety (e.g.an antibody) binds stronger to a target such as an epitope for which itis specific compared to the binding to another target. A binding moietybinds stronger to a first target compared to a second target if it bindsto the first target with a dissociation constant (K_(d)) which is lowerthan the dissociation constant for the second target. Preferably thedissociation constant (K_(d)) for the target to which the binding moietybinds specifically is more than 10-fold, preferably more than 20-fold,more preferably more than 50-fold, even more preferably more than100-fold, 200-fold, 500-fold or 1000-fold lower than the dissociationconstant (K_(d)) for the target to which the binding moiety does notbind specifically.

As used herein, the term “K_(d)” (measured in “mol/L”, sometimesabbreviated as “M”) is intended to refer to the dissociation equilibriumconstant of the particular interaction between a binding moiety (e.g. anantibody or fragment thereof) and a target molecule (e.g. an antigen orepitope thereof).

An “epitope”, also known as antigenic determinant, is the part of amacromolecule that is recognized by the immune system, specifically byantibodies, B cells, or T cells. As used herein, an “epitope” is thepart of a macromolecule capable of binding to a binding moiety (e.g. anantibody or antigen-binding fragment thereof) as described herein. Inthis context, the term “binding” preferably relates to a specificbinding. Epitopes usually consist of chemically active surface groupingsof molecules such as amino acids or sugar side chains and usually havespecific three-dimensional structural characteristics, as well asspecific charge characteristics. Conformational and non-conformationalepitopes are distinguished in that the binding to the former but not thelatter is lost in the presence of denaturing solvents.

EMBODIMENTS OF THE INVENTION

In the following several aspects of the invention are described in moredetail. In this context the meaning of certain terms is furtherexplained and preferred embodiments are indicated. These terms have thesame meaning for all aspects of the invention unless the content clearlydictates otherwise. Similarly, the preferred embodiments of one aspectthat relates to a similar subject-matter is also a preferred embodimentof another aspect.

In a first aspect the present invention provides a nucleic acidcomprising a nucleotide sequence encoding:

(a) a mammalian ErbB3 mutant protein comprising in comparison to therespective wildtype ErbB3 protein an amino acid substitution, whichchanges the conformation of the extracellular domain (ECD) of said ErbB3to an extended conformation;

(b) a N- and/or C-terminal deletion fragment of (a) comprising at leastthe ECD of the ErbB3 protein; or

(c) variant of (a) or (b), which has at least 80%, preferably at least85%, preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, more preferably at least 93%, more preferablyat least 94%, more preferably at least 95%, even more preferably atleast 96%, even more preferably at least 97%, even more preferably atleast 98%, even more preferably at least 99% or most preferably at least100% amino acid sequence identity to the amino acid of (a) or (b).

Preferably one or more of the nucleotide codons encoding (a), (b) or (c)that occur with low frequency in proteins expressed in mammalian cellshave been replaced with nucleotide codons that occur in nucleic acidsencoding highly expressed proteins.

The term “ErbB3 mutant protein”, as used herein, further refers to amutant protein comprising an amino acid mutation of ErbB3 protein incomparison to the respective wildtype ErbB3 protein, wherein the nucleicacid sequence of the ErbB3 mutant protein is shown in SEQ ID NO: 2,which changes the conformation of the extracellular domain (ECD) of saidErbB3 to an extended conformation. As used herein, the term“extracellular domain” (ECD) refers to the domain of the membrane-boundreceptor ErbB3 which sticks out of the membrane in to the cytoplasm andtherefore on the outside of the cell.

In a preferred embodiment of the first aspect an ErbB3 protein is from amammal, preferably from rat (Accession No., AAC53050.1), mouse(Accession No., AAA93533.1) or human (Accession No., AAH02706.1), mostpreferably from human.

It is also preferred that the mutation changes the conformation of theextracellular domain (ECD) of said ErbB3, most preferred to an extendedconformation as described in [22]. The conformational change results ina better binding to a ligand, e.g. neuregulin or other ligands which canbind to the ECD of ErbB3 and thus, trigger the signal transduction ofErbB3. The ECD of ErbB3 spans amino acids 20 to 643 according to SEQ IDNO: 2 or amino acids occupying analogous positions in another ErbB3.

It is further preferred that the nucleic acid comprising a nucleotidesequence encoding a deletion fragment of at least the ECD of the ErbB3protein is the nucleic acid according to SEQ ID NO: 3.

In a preferred embodiment of the nucleic acid of the present inventionthe following nucleotide codons that occur with low frequency inexpressed proteins are replaced with the following nucleotide codonsthat occur in nucleic acids encoding highly expressed proteins. Thisfeature of the nucleic acid of the invention can alternatively bereferred to as codon optimization for expression in mammalian,preferably human cells. Thus, in a preferred embodiment the nucleic acidof the invention is codon optimized for expression in mammalian cells,in particular in humans. In the following a codon usage table forproteins in human cells is indicated.

Codon Usage Table fields: [frequency: per thousand]([number]) as determined for 93487 coding sequences UUU 17.6(714298)UCU 15.2(618711) UAU 12.2(495699) UGU 10.6(430311) UUC 20.3(824692)UCC 17.7(718892) UAC 15.3(622407) UGC 12.6(513028) UUA  7.7(311881)UCA 12.2(496448) UAA  1.0(40285) UGA  1.6(63237) UUG 12.9(525688)UCG  4.4(179419) UAG  0.8(32109) UGG 13.2(535595) CUU 13.2(536515)CCU 17.5(713233) CAU 10.9(441711) CGU  4.5(184609) CUC 19.6(796638)CCC 19.8(804620) CAC 15.1(613713) CGC 10.4(423516) CUA  7.2(290751)CCA 16.9(688038) CAA 12.3(501911) CGA  6.2(250760) CUG 39.6(1611801)CCG  6.9(281570) CAG 34.2(1391973) CGG 11.4(464485) AUU 16.0(650473)ACU 13.1(533609) AAU 17.0(689701) AGU 12.1(493429) AUG 20.8(846466)ACC 18.9(768147) AAC 19.1(776603) AGC 19.5(791383) AUA   7.5(304565)ACA 15.1(614523) AAA 24.4(993621) AGA 12.2(494682) AUG 22.0(896005)ACG  6.1(246105) AAG 31.9(1295568) AGG 12.0(486463) GUU 11.0(448607)GCU 18.4(750096) GAU 21.8(885429) GGU 10.8(437126) GUC 14.5(588138)GCC 27.7(1127679) GAC 25.1(1020595) GGC 22.2(903565) GUA  7.1(287712)GCA 15.8(643471) GAA 29.0(1177632) GGA 16.5(669873) GUG 28.1(1143534)GCG  7.4(299495) GAG 39.6(1609975) GGG 16.5(669768)These numbers translate to the following preferred replacements of lowor lower frequency codons, with higher or highest frequency codons. Themost preferred replacements are highlighted:

Encoded Low or lower Amino Frequency Codon Higher or highest Acidto be replaced Frequency Codon Phe UUU 17.6(714298) UUC 20.3(824692) LeuCUA  7.2(290751) UUA  7.7(311881) Leu CUA  7.2(290751) UUG 12.9(525688)Leu CUA  7.2(290751) CUU 13.2(536515) Leu CUA  7.2(290751)CUC 19.6(796638) Leu CUA  7.2(290751) CUG 39.6(1611801) LeuUUA  7.7(311881) UUG 12.9(525688) Leu UUA  7.7(311881) CUU 13.2(536515)Leu UUA  7.7(311881) CUC 19.6(796638) Leu UUA  7.7(311881)CUG 39.6(1611801) Leu UUG 12.9(525688) CUU 13.2(536515) LeuUUG 12.9(525688) CUC 19.6(796638) Leu UUG 12.9(525688) CUG 39.6(1611801)Leu CUU 13.2(536515) CUC 19.6(796638) Leu CUU 13.2(536515)CUG 39.6(1611801) Leu CUC 19.6(796638) CUG 39.6(1611801) IleAUA  7.5(304565) AUU 16.0(650473) Ile AUA  7.5(304565) AUC 20.8(846466)Ile AUU 16.0(650473) AUC 20.8(846466) Val GUA  7.1(287712)GUU 11.0(448607) Val GUA  7.1(287712) GUC 14.5(588138) ValGUA  7.1(287712) GUG 28.1(1143534) Val GUU 11.0(448607) GUC 14.5(588138)Val GUU 11.0(448607) GUG 28.1(1143534) Val GUC 14.5(588138)GUG 28.1(1143534) Ser UCG  4.4(179419) AGU 12.1(493429) SerUCG  4.4(179419) UCA 12.2(496448) Ser UCG  4.4(179419) UCU 15.2(618711)Ser UCG  4.4(179419) UCC 17.7(718892) Ser UCG  4.4(179419)AGC 19.5(791383) Ser AGU 12.1(493429 UCA 12.2(496448) SerAGU 12.1(493429 UCU 15.2(618711) Ser AGU 12.1(493429 UCC 17.7(718892)Ser AGU 12.1(493429 AGC 19.5(791383) Ser UCA 12.2(496448)UCU 15.2(618711) Ser UCA 12.2(496448) UCC 17.7(718892) SerUCA 12.2(496448) AGC 19.5(791383) Ser UCU 15.2(618711) UCC 17.7(718892)Ser UCU 15.2(618711) AGC 19.5(791383) Ser UCC 17.7(718892)AGC 19.5(791383) Pro CCG  6.9(281570) CCA 16.9(688038) ProCCG  6.9(281570) CCU 17.5(713233) Pro CCG  6.9(281570) CCC 19.8(804620)Pro CCA 16.9(688038) CCU 17.5(713233) Pro CCA 16.9(688038)CCC 19.8(804620) Pro CCU 17.5(713233) CCC 19.8(804620) ThrACG  6.1(246105) ACU 13.1(533609) Thr ACG  6.1(246105) ACA 15.1(614523)Thr ACG  6.1(246105) ACC 18.9(768147) Thr ACU 13.1(533609)ACA 15.1(614523) Thr ACU 13.1(533609) ACC 18.9(768147) ThrACA 15.1(614523) ACC 18.9(768147) Ala GCG  7.4(299495) GCA 15.8(643471)Ala GCG  7.4(299495) GCU 18.4(750096) Ala GCG  7.4(299495)GCC 27.7(1127679) Ala GCU 15.8(643471) GCU 18.4(750096) AlaGCU 15.8(643471) GCC 27.7(1127679) Ala GCU 18.4(750096)GCC 27.7(1127679) Tyr UAU 12.2(495699) UAC 15.3(622407) StoppUAG  0.8(32109) UAA  1.0(40285) Stopp UAG  0.8(32109) UGA  1.6(63237)Stopp UAA  1.0(40285) UGA  1.6(63237) His CAU 10.9(441711)CAC 15.1(613713) Gln CAA 12.3(501911) CAG 34.2(1391973) AsnAAU 17.0(689701) AAC 19.1(776603) Lys AAA 24.4(993621) AAG 31.9(1295568)Asp GAU 21.8(885429) GAC 25.1(1020595) Glu GAA 29.0(1177632)GAG 39.6(1609975) Cys UGU 10.6(430311) UGC 12.6(513028) ArgCGU  4.5(184609) CGA  6.2(250760) Arg CGU  4.5(184609) CGC 10.4(423516)Arg CGU  4.5(184609) CGG 11.4(464485) Arg CGU  4.5(184609)AGG 12.0(486463) Arg CGU  4.5(184609) AGA 12.2(494682) ArgCGA  6.2(250760) CGC 10.4(423516) Arg CGA  6.2(250760) CGG 11.4(464485)Arg CGA  6.2(250760) AGG 12.0(486463) Arg CGA  6.2(250760)AGA 12.2(494682) Arg CGC 10.4(423516) CGG 11.4(464485) ArgCGC 10.4(423516) AGG 12.0(486463) Arg CGC 10.4(423516) AGA 12.2(494682)Arg CGG 11.4(464485) AGG 12.0(486463) Arg CGG 11.4(464485)AGA 12.2(494682) Arg AGG 12.0(486463) AGA 12.2(494682) GlyGGU 10.8(437126) GGG 16.5(669768) Gly GGU 10.8(437126) GGA 16.5(669873)Gly GGU 10.8(437126) GGC 22.2(903565) Gly GGG 16.5(669768)GGA 16.5(669873) Gly GGG 16.5(669768) GGC 22.2(903565) GlyGGA 16.5(669873) GGC 22.2(903565)

It is preferred that at least 10% of all low or lower frequency codonsare replaced by high or higher frequency codons, more preferably atleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In a particularpreferred embodiment all low or lower frequency codons are replaced withthe respective highest frequency codon encoding a given amino acid. Theresulting nucleic acid, thus, differs from the naturally occurringnucleic acid but encodes the identical protein but for the substitutiondiscussed above.

In a preferred embodiment of the first aspect of the present invention,the amino acid at position 584 according to SEQ ID NO: 2 or at an aminoacid occupying an analogous position is mutated to another amino acid,preferably to Gly, Ala, Val, Cys, Arg, Pro, Ser, Leu, Ile, Met, Tyr,Thr, Trp, Gln, Asn, Asp, Glu, Lys, or Phe, most preferably to Phe

It is also preferred that the encoded polypeptide is selected from thegroup consisting of SEQ ID NO: 2 and SEQ ID NO: 4.

In a second aspect the present invention is directed to an expressionvector comprising the nucleic acid of the first aspect of the invention,operatively linked to a promoter or to a regulatory transcriptionalelement. For example, to a promoter which is a regulatory region of theDNA which is located upstream (towards the 5′ region of the sensestrand) of a gene, facilitating the transcription of a gene. Thestructure of gene promoters can be quite complex, typically consistingof multiple transcriptional regulatory elements, for example corepromoters, proximal promoters, distal enhancers, silencers,insulators/boundary elements, for further information see Maston et al.[34].

In a preferred embodiment, the expression vector is selected from thegroup consisting of a bacterial plasmid, an adenovirus, a poxvirus, avaccinia virus, a fowlpox virus, a herpes virus, an adeno-associatedvirus (AAV), an alphavirus, a lentivirus, a lambda phage, a lymphocyticchoriomeningitis virus and a Listeria sp, Salmonella sp.

In a third aspect the present invention is directed to a nucleic acid ofthe first aspect or an expression vector of the second aspect for use inpreventing, treating or delaying neoplasms in a mammal. Thus, thepresent invention relates to a method of preventing, treating ordelaying neoplasm in a mammal, wherein an effective amount of saidnucleic acid or expression vector is administered to a subject in needthereof. Neoplasm is uncontrolled cell growth as a result of neoplasiawhich means abnormal proliferation of cells. Neoplasm can cause a tumorand/or cancer.

In a further embodiment of this aspect the present invention is directedto a nucleic acid or an expression vector, wherein one or more of thenucleotide codons of the nucleotide encoding the protein according to(a), (b) or (c) is a nucleotide codon that occurs with low frequency inproteins expressed in mammalian cells have been replaced with nucleotidecodons that occur in nucleic acids encoding highly expressed proteins.

In a further embodiment of this aspect an immune response, preferably aT and/or B cell response is generated against said neoplasm.

In a further embodiment of this aspect the mammal is a human, mouse,rat, dog, cat, horse, more preferably human, mouse, rat or dog, mostpreferred human.

In a preferred embodiment of this aspect the nucleic acid comprises anucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

It is preferred that the nucleic acid or expression vector isadministered parenteral. Parenteral administration preferably comprisesintramuscular, subcutan, intradermal, intra-arterial, intrasternal,intracranial, intrathoracic, intraspinal and/or into the neoplasm insitu. The most preferred administration is intramuscular.

It is preferred that the administration further comprises the step ofapplying electroporation, preferably to the site of administration ofthe nucleic acid or expression vector.

It is also preferred to administering an immune response potentiator tothe mammal. This may be administered prior, concomittantly or afteradministration of the nucleic acid or expression vector of theinvention. For administration the nucleic acid or expression vector canbe combined with one or more adjuvants for the route of administration,e.g. dissolved in saline.

Preferably, the nucleic acid or the expression vector is co-administeredwith an anti-neoplastic agent or antineoplatic regimen. Preferably, theanti-neoplastic agent is selected from the group consisting of ananti-angiogenic agent, an alkylating agent, an antimetabolite, a naturalproduct, a platinum coordination complex, an anthracenedione, asubstituted urea, a methylhydrazine derivative, an adrenocorticalsuppressant, a hormone, an antagonist, an oncogene inhibitor, a tumorsuppressor gene or protein, a therapeutic antibody and an anti-oncogeneoligonucleotide.

Neoplasm to be prevented, treated or delayed is selected from the groupconsisting of adrenal gland, anus, auditory nerve, bile ducts, bladder,bone, brain, breast, central nervous system, cervix, colon, ear,endometrium, esophagus, eye, eyelids, fallopian tube, gastrointestinaltract, head and neck, heart, kidney, larynx, liver, lung, mandible,mandibular condyle, maxilla, mouth, nasopharinx, nose, oral cavity,ovary, pancreas, parotid gland, penis, pinna, pituitary, prostate gland,rectum, retina, salivary glands, skin, small intestine, spinal cord,stomach, testes, thyroid, tonsil, urethra, uterus, vagina,vestibulocochlear nerve and vulva neoplasm, preferably breast cancer,preferably lung cancer, preferably pancreatic cancer, preferably ovariancancer, preferably gastric cancer, preferably prostate cancer ormelanoma.

In a fourth aspect the present invention is directed to a method ofgenerating an antibody against mammalian ErbB3 comprising the step ofadministering to a mammal:

-   -   (a) a mammalian ErbB3 mutant protein comprising in comparison to        the respective wildtype ErbB3 protein an amino acid        substitution, which changes the conformation of the        extracellular domain (ECD) of said ErbB3 to an extended        conformation,    -   (b) a N- and/or C-terminal deletion fragment of (a) comprising        at least the ECD of the ErbB3 protein; or    -   (c) a variant of (a) or (b), which has at least 80%, preferably        at least 85%, preferably at least 90%, more preferably at least        91%, more preferably at least 92%, more preferably at least 93%,        more preferably at least 94%, more preferably at least 95%, even        more preferably at least 96%, even more preferably at least 97%,        even more preferably at least 98%, even more preferably at least        99% or most preferably at least 100% amino acid sequence        identity to the amino acid of (a) or (b) or a nucleotide        encoding the protein according to (a), (b) or (c).

It is preferred in this method that one or more of the nucleotide codonsof the nucleotide encoding the protein according to (a), (b) or (c) is anucleotide codon that occurs with low frequency in proteins expressed inmammalian cells have been replaced with nucleotide codons that occur innucleic acids encoding highly expressed proteins.

In a fifth aspect the present invention relates to an antibody producedor producible according to the method of the present invention, whereinthe antibody binds to an epitope in an extracellular domain of the ErbB3protein, or a functional fragment thereof and exhibits one or more ofthe following properties: i. inhibition of heregulin, epiregulin,betacellulin, epigen or biregulin-mediated signalling through ErbB3which can be measured according to the methods of the present invention;ii. inhibition of proliferation of cells expressing ErbB3 which can bemeasured according to the methods of the present invention or othermethods which are well known in the art for the skilled person (e.g.cell proliferation assays which can be obtained by Invitrogen, Promegaor other chemical companies familiar to those skilled in the art); iii.the ability to decrease levels of ErbB3 on cell surfaces which can bemeasured according to the methods of the present invention; iv.inhibition of VEGF secretion of cells expressing ErbB3 which can bemeasured according to the methods of the present invention or othermethods which are well-known for the skilled person (e.g. ELISA assaywhich can be obtained from Millipore or other chemical companiesfamiliar to those skilled in the art); v. inhibition of the migration ofcells expressing ErbB3 which can be measured according to the methods ofthe present invention or migration assay which are well-known for theskilled person (see e.g. migration assays which can be obtained fromMillipore or other chemical companies familiar to those skilled in theart); vi. inhibition of spheroid growth of cells expressing ErbB3 whichcan be measured according to the methods of the present invention; vii.a binding specificity to ErbB3 of 20 nM or less, preferably 10 nM orless more preferably 8 nM or less which can be measured according to themethods of the present invention other methods which are well known inthe art for the skilled person, e.g. by SPR on a biacore (sodium plasmonresonance on a biacore, see e.g., Maier S. A. et al. [45]); or viii.inhibits homo- and/or heterodimerization of ErbB3.

Preferably the antibody of the invention comprises the followingproperties (i) and (ii); (i), (ii) and (iii); (i), (ii), (iii) and (iv);(i), (ii), (iii), (iv), and (v); (i), (ii), (iii), (iv), (v), and (vi);(i), (ii), (iii), (iv), (v), (vi), and (vii); (i), (ii), (iii), (iv),(v), (vi), (vii), and (viii); (ii), (iii), (iv), (v), (vi), (vii), and(viii); (iii), (iv), (v), (vi), (vii), and (viii); (iv), (v), (vi),(vii), and (viii); (vi), (vii), and (viii); (vii), and (viii); (i),(iii), (iv), (v), (vi), (vii), and (viii); (i), (iv), (v), (vi), (vii),and (viii); (i), (v), (vi), (vii), and (viii); (i), (vii), and (viii);(i), and (viii); (i), (ii), (iv), (v), (vi), (vii), and (viii); (i),(ii), (v), (vi), (vii), and (viii); (i), (ii), (vi), (vii), and (viii);(i), (ii), (vii), and (viii); (i), (ii), and (viii); (i), (ii), (iii),(v), (vi), (vii), and (viii); (i), (ii), (iii), (vi), (vii), and (viii);(i), (ii), (iii), (vii), and (viii); (i), (ii), (iii), and (viii); (i),(ii), (iii), (iv), (vi), (vii), and (viii); (i), (ii), (iii), (iv),(vii), and (viii); (i), (ii), (iii), (iv), and (viii); (i), (ii), (iii),(iv), (v), (vii), and (viii); (i), (ii), (iii), (iv), (v), and (viii);(i), (ii), (iii), (iv), (v), (vi), and (viii); (ii), (iv), (v), (vi),(vii), and (viii); (ii), (v), (vi), (vii), and (viii); (ii), (vi),(vii), and (viii); (ii), (vii), and (viii); (ii), and (viii); (ii),(iii), (v), (vi), (vii), and (viii); (ii), (iii), (vi), (vii), and(viii); (ii), (iii), (vii), and (viii); (ii), (iii), and (viii); (ii),(iii), (iv), (vi), (vii), and (viii); (ii), (iii), (iv), (vii), and(viii); (ii), (iii), (iv), and (viii); or (ii), (iii), (iv), (v), (vii),and (viii); (ii), (iii), (iv), (v), (viii).

In a preferred embodiment the antibody of the present inventionspecifically binds to:

(i) an epitope of human ErbB3 which is formed by the amino acidsequences spanning positions 215 to 227 of human ErbB3 according to SEQID NO: 2,

(ii) to an epitope that occupies analogous positions to amino acids 215to 227 of SEQ ID NO: 2 in another ErbB3 protein

(iii) or an at least 8 amino acid long fragment of (i) or (ii). In thiscontext the term “specifically binds to” refers to a binding specificityof at least 1000 nM, preferably 900 nM or less, more preferably 800 nMor less, more preferably 700 nM or less, more preferably 600 nM or less,more preferably 500 nM or less, more preferably 400 nM or less, morepreferably 300 nM or less, more preferably 200 nM or less, even morepreferably 100 nM or less, even more preferably 90 nM or less, even morepreferably 80 nM or less, even more preferably 70 nM or less, even morepreferably 60 nM or less, even more preferably 50 nM or less, even morepreferably 40 nM or less, even more preferably 30 nM or less, even morepreferably 20 nM or less, even more preferably 10 nM or less, even morepreferably 8 nM or less, most preferably 8 nM or less.

Preferably the epitope of ErbB3 is an at least 8 amino acid longfragment comprising the following amino acids QCNGHCFG (SEQ ID NO: 33),CNGHCFGP (SEQ ID NO: 34), NGHCFGPN (SEQ ID NO: 35), GHCFGPNP (SEQ ID NO:36), HCFGPNPN (SEQ ID NO: 37), CFGPNPNQ (SEQ ID NO: 38), FGPNPNQC (SEQID NO: 39) or GPNPNQCC (SEQ ID NO: 40), preferably the epitope of ErbB3is an at least 9 amino acid long fragment comprising the following aminoacids QCNGHCFGP (SEQ ID NO: 41), CNGHCFGPN (SEQ ID NO: 42), NGHCFGPNP(SEQ ID NO: 43), GHCFGPNPN (SEQ ID NO: 44), HCFGPNPNQ (SEQ ID NO: 45),CFGPNPNQC (SEQ ID NO: 46) or FGPNPNQCC (SEQ ID NO: 47), preferably theepitope of ErbB3 is an at least 10 amino acid long fragment comprisingthe following amino acids QCNGHCFGPN (SEQ ID NO: 48), CNGHCFGPNP (SEQ IDNO: 49), NGHCFGPNPN (SEQ ID NO: 50), GHCFGPNPNQ (SEQ ID NO: 51),HCFGPNPNQC (SEQ ID NO: 52) or CFGPNPNQCC (SEQ ID NO: 53), preferably theepitope of ErbB3 is an at least 11 amino acid long fragment comprisingthe following amino acids QCNGHCFGPNP (SEQ ID NO: 54), CNGHCFGPNPN (SEQID NO: 55), NGHCFGPNPNQ (SEQ ID NO: 56), GHCFGPNPNQC (SEQ ID NO: 57) orHCFGPNPNQCC (SEQ ID NO: 58), preferably the epitope of ErbB3 is an atleast 12 amino acid long fragment comprising the following amino acidsQCNGHCFGPNPN (SEQ ID NO: 59), CNGHCFGPNPNQ (SEQ ID NO: 60), NGHCFGPNPNQC(SEQ ID NO: 61) or GHCFGPNPNQCC (SEQ ID NO: 62), preferably the epitopeof ErbB3 is an at least 13 amino acid long fragment comprising thefollowing amino acids QCNGHCFGPNPNQ (SEQ ID NO: 63), CNGHCFGPNPNQC (SEQID NO: 64) or NGHCFGPNPNQCC (SEQ ID NO: 65), preferably the epitope ofErbB3 is an at least 14 amino acid long fragment comprising thefollowing amino acids CNGHCFGPNPNQCC (SEQ ID NO: 66) or QCNGHCFGPNPNQC(SEQ ID NO: 67), preferably the epitope of ErbB3 is an at least 15 aminoacid long fragment comprising the following amino acids QCNGHCFGPNPNQCC(SEQ ID NO: 68), most preferably the epitope of ErbB3 is an at least 15amino acid long fragment comprising the following amino acidsQCNGHCFGPNPNQCC (SEQ ID NO: 68). This epitope spans amino acids 215 to227 of human ErbB3 according to SEQ ID NO: 2. Alternatively, the ErbB3epitope to which the antibodies of the invention bind is an epitope thatoccupies analogous positions to amino acids 215 to 227 of SEQ ID NO: 2in another ErbB3 protein.

In a preferred embodiment the antibody of present invention comprises:(i) a light chain CDR3 sequence as set forth in SEQ ID NO: 19 or asequence, which comprises with respect to SEQ ID NO: 19 one or two aminoacid substitutions, deletions and/or insertions, preferably one or twoamino acid substitutions; or (ii) a light chain CDR3 sequence as setforth in SEQ ID NO: 25 or a sequence, which comprises with respect toSEQ ID NO: 25 one or two amino acid substitutions, deletions and/orinsertions, preferably one or two amino acid substitutions. In thiscontext it is preferred that the antibody or antigen-binding fragmentthereof further comprises: (i) a light chain CDR1 sequence according toSEQ ID NO: 17 or a sequence, which comprises with respect to SEQ ID NO:17 one, two or three amino acid substitutions, deletions and/orinsertions, preferably one, two or three amino acid substitutions; (ii)a light chain CDR1 sequence according to SEQ ID NO: 23 or a sequence,which comprises with respect to SEQ ID NO: 23 one, two or three aminoacid substitutions, deletions and/or insertions, preferably one, two orthree amino acid substitutions; (iii) a light chain CDR2 sequenceaccording to SEQ ID NO: 18 or a sequence, which comprises with respectto SEQ ID NO: 18 one or two amino acid substitutions, deletions and/orinsertions, preferably one or two amino acid substitutions; (iv) a lightchain CDR2 sequence according to SEQ ID NO: 24 or a sequence, whichcomprises with respect to SEQ ID NO: 24 one or two amino acidsubstitutions, deletions and/or insertions, preferably one or two aminoacid substitutions; (v) a heavy chain CDR1 sequence according to SEQ IDNO: 20 or a sequence, which comprises with respect to SEQ ID NO: 20 one,two or three amino acid substitutions, deletions and/or insertions,preferably one, two or three amino acid substitutions; (vi) a heavychain CDR1 sequence according to SEQ ID NO: 26 or a sequence, whichcomprises with respect to SEQ ID NO: 26 one, two or three amino acidsubstitutions, deletions and/or insertions, preferably one, two or threeamino acid substitutions; (vii) a heavy chain CDR2 sequence according toSEQ ID NO: 21 or a sequence, which comprises with respect to SEQ ID NO:21 one or two amino acid substitutions, deletions and/or insertions,preferably one or two amino acid substitutions; (viii) a heavy chainCDR2 sequence according to SEQ ID NO: 27 or a sequence, which compriseswith respect to SEQ ID NO: 27 one or two amino acid substitutions,deletions and/or insertions, preferably one or two amino acidsubstitutions; a heavy chain CDR3 sequence according to SEQ ID NO: 22 ora sequence, which comprises with respect to SEQ ID NO: 22 one or twoamino acid substitutions, deletions and/or insertions; preferably one ortwo amino acid substitutions; (x) a heavy chain CDR3 sequence accordingto SEQ ID NO: 28 or a sequence, which comprises with respect to SEQ IDNO: 28 one or two amino acid substitutions, deletions and/or insertions,preferably one or two amino acid substitutions; (xi) a light chain CDR1sequence according to SEQ ID NO: 29 or a sequence, which comprises withrespect to SEQ ID NO: 29 one, two or three amino acid substitutions,deletions and/or insertions, preferably one, two or three amino acidsubstitutions; (xii) a light chain CDR2 sequence according to SEQ ID NO:30 or a sequence, which comprises with respect to SEQ ID NO: 30 one ortwo amino acid substitutions, deletions and/or insertions, preferablyone or two amino acid substitutions; (xiii) a heavy chain CDR1 sequenceaccording to SEQ ID NO: 31 or a sequence, which comprises with respectto SEQ ID NO: 31 one, two or three amino acid substitutions, deletionsand/or insertions, preferably one, two or three amino acidsubstitutions; (xiv) a heavy chain CDR2 sequence according to SEQ ID NO:32 or a sequence, which comprises with respect to SEQ ID NO: 32 one ortwo amino acid substitutions, deletions and/or insertions, preferablyone or two amino acid substitutions.

In a preferred embodiment the antibody or an antigen-binding fragmentthereof comprises: (a) a heavy chain CDR3 sequence as set forth in SEQID NO: 22 or a sequence, which comprises with respect to SEQ ID NO: 22one or two amino acid substitutions, deletions and/or insertions,preferably one or two amino acid substitutions; or (b) a heavy chainCDR3 sequence as set forth in SEQ ID NO: 28 or a sequence, whichcomprises with respect to SEQ ID NO: 28 one or two amino acidsubstitutions, deletions and/or insertions, preferably one or two aminoacid substitutions. In this context it is preferred that the antibody orantigen-binding fragment thereof further comprises: (a) a light chainCDR1 sequence according to SEQ ID NO: 17 or a sequence, comprises withrespect to SEQ ID NO: 17 one, two or three amino acid substitutions,deletions and/or insertions, preferably one, two or three amino acidsubstitutions; (b) a light chain CDR1 sequence according to SEQ ID NO:23 or a sequence, which comprises with respect to SEQ ID NO: 23 one, twoor three amino acid substitutions, deletions and/or insertions,preferably one, two or three amino acid substitutions; (c) a light chainCDR2 sequence according to SEQ ID NO: 18 or a sequence, which compriseswith respect to SEQ ID NO: 18 one or two amino acid substitutions,deletions and/or insertions, preferably one or two amino acidsubstitutions; (d) a light chain CDR2 sequence according to SEQ ID NO:24 or a sequence, which comprises with respect to SEQ ID NO: 24 one ortwo amino acid substitutions, deletions and/or insertions, preferablyone or two amino acid substitutions; (e) a light chain CDR3 sequenceaccording to SEQ ID NO: 19 or a sequence, which comprises with respectto SEQ ID NO: 19 one or two amino acid substitutions, deletions and/orinsertions, preferably one or two amino acid substitutions; (f) a lightchain CDR3 sequence according to SEQ ID NO: 25 or a sequence, whichcomprises with respect to SEQ ID NO: 25 one or two amino acidsubstitutions, deletions and/or insertions, preferably one or two aminoacid substitutions; (g) a heavy chain CDR1 sequence according to SEQ IDNO: 20 or a sequence, which comprises with respect to SEQ ID NO: 20 one,two or three amino acid substitutions, deletions and/or insertions,preferably one, two or three amino acid substitutions; (h) a heavy chainCDR1 sequence according to SEQ ID NO: 26 or a sequence, which compriseswith respect to SEQ ID NO: 26 one, two or three amino acidsubstitutions, deletions and/or insertions, preferably one, two or threeamino acid substitutions; (i) a heavy chain CDR2 sequence according toSEQ ID NO: 21 or a sequence, which comprises with respect to SEQ ID NO:21 one or two amino acid substitutions, deletions and/or insertions,preferably one or two amino acid substitutions; (j) a heavy chain CDR2sequence according to SEQ ID NO: 27 or a sequence, which comprises withrespect to SEQ ID NO: 27 one or two amino acid substitutions, deletionsand/or insertions, preferably one or two amino acid substitutions; (k) alight chain CDR1 sequence according to SEQ ID NO: 29 or a sequence,which comprises with respect to SEQ ID NO: 29 one, two or three aminoacid substitutions, deletions and/or insertions, preferably one, two orthree amino acid substitutions; (1) a light chain CDR2 sequenceaccording to SEQ ID NO: 30 or a sequence, which comprises with respectto SEQ ID NO: 30 one or two amino acid substitutions, deletions and/orinsertions, preferably one or two amino acid substitutions; (m) a heavychain CDR1 sequence according to SEQ ID NO: 31 or a sequence, whichcomprises with respect to SEQ ID NO: 31 one, two or three amino acidsubstitutions, deletions and/or insertions, preferably one, two or threeamino acid substitutions; (n) a heavy chain CDR2 sequence according toSEQ ID NO: 32 or a sequence, which comprises with respect to SEQ ID NO:32 one or two amino acid substitutions, deletions and/or insertions,preferably one or two amino acid substitutions.

In a preferred embodiment of the antibody of the invention orantigen-binding fragment thereof it comprises one of the sets of heavychain CDR3, heavy chain CDR2, and heavy chain CDR1 sequences as listedbelow in Table 1, wherein each heavy chain CDR3 sequence comprises oneor two amino acid substitutions, deletions and/or insertions, preferablyone or two amino acid substitutions as listed in Table 1; wherein eachheavy chain CDR2 sequence comprises one or two amino acid substitutions,deletions and/or insertions, preferably one or two amino acidsubstitutions as listed in Table 1; and wherein each heavy chain CDR1sequence comprises with respect to SEQ ID NO: 30 one, two or three aminoacid substitutions, deletions and/or insertions, preferably one, two orthree amino acid substitutions as listed in Table 1.

TABLE 1 Sets of heavy chain CDR sequences suitable for use in theantibodies or fragments thereof of the present invention Symbol of heavychain set CDR3 sequence CDR2 sequence CDR1 sequence A SEQ ID NO: 22 SEQID NO: 21 SEQ ID NO: 20 B SEQ ID NO: 22 SEQ ID NO: 21 SEQ ID NO: 26 CSEQ ID NO: 22 SEQ ID NO: 27 SEQ ID NO: 26 D SEQ ID NO: 22 SEQ ID NO: 27SEQ ID NO: 20 E SEQ ID NO: 28 SEQ ID NO: 21 SEQ ID NO: 20 F SEQ ID NO:28 SEQ ID NO: 27 SEQ ID NO: 20 G SEQ ID NO: 28 SEQ ID NO: 27 SEQ ID NO:26 H SEQ ID NO: 28 SEQ ID NO: 21 SEQ ID NO: 26 I SEQ ID NO: 22 SEQ IDNO: 21 SEQ ID NO: 31 J SEQ ID NO: 22 SEQ ID NO: 32 SEQ ID NO: 31 K SEQID NO: 22 SEQ ID NO: 32 SEQ ID NO: 20 L SEQ ID NO: 28 SEQ ID NO: 32 SEQID NO: 31 M SEQ ID NO: 28 SEQ ID NO: 27 SEQ ID NO: 31 N SEQ ID NO: 28SEQ ID NO: 32 SEQ ID NO: 26 O SEQ ID NO: 22 SEQ ID NO: 27 SEQ ID NO: 31P SEQ ID NO: 28 SEQ ID NO: 21 SEQ ID NO: 31 Q SEQ ID NO: 22 SEQ ID NO:32 SEQ ID NO: 26 R SEQ ID NO: 28 SEQ ID NO: 32 SEQ ID NO: 20

In a preferred embodiment of the antibody of the invention orantigen-binding fragment thereof it comprises one of the sets of lightchain CDR3, light chain CDR2, and light chain CDR1 sequences as listedbelow in Table 2, wherein each light chain CDR3 sequence comprises oneor two amino acid substitutions, deletions and/or insertions, preferablyone or two amino acid substitutions as listed in Table 2; wherein eachlight chain CDR2 sequence comprises one or two amino acid substitutions,deletions and/or insertions, preferably one or two amino acidsubstitutions as listed in Table 2; and wherein each light chain CDR1sequence comprises one, two or three amino acid substitutions, deletionsand/or insertions, preferably one, two or three amino acid substitutionsas listed in Table 2.

TABLE 2 Sets of light chain CDR sequences suitable for use in theantibodies or fragments thereof of the present invention Symbol of lightchain set CDR3 sequence CDR2 sequence CDR1 sequence I SEQ ID NO: 19 SEQID NO: 18 SEQ ID NO: 17 II SEQ ID NO: 19 SEQ ID NO: 18 SEQ ID NO: 23 IIISEQ ID NO: 19 SEQ ID NO: 24 SEQ ID NO: 23 IV SEQ ID NO: 19 SEQ ID NO: 24SEQ ID NO: 17 V SEQ ID NO: 25 SEQ ID NO: 18 SEQ ID NO: 17 VI SEQ ID NO:25 SEQ ID NO: 24 SEQ ID NO: 17 VII SEQ ID NO: 25 SEQ ID NO: 24 SEQ IDNO: 23 VIII SEQ ID NO: 25 SEQ ID NO: 18 SEQ ID NO: 23 IX SEQ ID NO: 19SEQ ID NO: 18 SEQ ID NO: 29 X SEQ ID NO: 19 SEQ ID NO: 30 SEQ ID NO: 29XI SEQ ID NO: 19 SEQ ID NO: 30 SEQ ID NO: 17 XII SEQ ID NO: 25 SEQ IDNO: 30 SEQ ID NO: 29 XIII SEQ ID NO: 25 SEQ ID NO: 24 SEQ ID NO: 29 XIVSEQ ID NO: 25 SEQ ID NO: 30 SEQ ID NO: 23 XV SEQ ID NO: 19 SEQ ID NO: 24SEQ ID NO: 29 XVI SEQ ID NO: 25 SEQ ID NO: 18 SEQ ID NO: 29 XVII SEQ IDNO: 19 SEQ ID NO: 30 SEQ ID NO: 23 XVIII SEQ ID NO: 25 SEQ ID NO: 30 SEQID NO: 17

In a preferred embodiment of the antibody of the invention or ofantigen-binding fragments thereof it comprises one of the heavy CDR setsA-R listed above in Table 1 and one of the light chain CDR sets I-XVIIIlisted above in Table 2, i.e. one of the following combinations of sets:A-I, A-II, A-III, A-IV, A-V, A-VI, A-VII, A-VIII, A-IX, A-X, A-XI,A-XII, A-XIII, A-XIV, A-XV, A-XVI, A-XVII, A-XVIII, B-I, B-II, B-III,B-IV, B-V, B-VI, B-VII, B-VIII, B-IX, B-X, B-XI, B-XII, B-XIII, B-XIV,B-XV, B-XVI, B-XVII, B-XVIII, C-I, C-II, C-III, C-IV, C-V, C-VI, C-VII,C-VIII, C-IX, C-X, C-XI, C-XII, C-XIII, C-XIV, C-XV, C-XVI, C-XVII,C-XVIII, D-I, D-II, D-III, D-IV, D-V, D-VI, D-VII, D-VIII, D-IX, D-X,D-XI, D-XII, D-XIII, D-XIV, D-XV, D-XVI, D-XVII, D-XVIII, E-I, E-II,E-III, E-IV, E-V, E-VI, E-VII, E-VIII, E-IX, E-X, E-XI, E-XII, E-XIII,E-XIV, E-XV, E-XVI, E-XVII, E-XVIII, F-I, F-II, F-III, F-IV, F-V, F-VI,F-VII, F-VIII, F-IX, F-X, F-XI, F-XII, F-XIII, F-XIV, F-XV, F-XVI,F-XVII, F-XVIII, G-I, G-IT, G-III, G-IV, G-V, G-VI, G-VII, G-VIII, G-IX,G-X, G-XI, G-XII, G-XIII, G-XIV, G-XV, G-XVI, G-XVII, G-XVIII, H-I,H-II, H-III, H-IV, H-V, H-VI, H-VII, H-VIII, H-IX, H-X, H-XI, H-XII,H-XIII, H-XIV, H-XV, H-XVI, H-XVII, H-XVIII, I-I, I-II, I-III, I-IV,I-V, I-VI, I-VII, I-VIII, I-IX, I-X, I-XI, I-XII, I-XIII, I-XIV, I-XV,I-XVI, I-XVII, I-XVIII, J-I, J-II, J-III, J-IV, J-V, J-VI, J-VII,J-VIII, J-IX, J-X, J-XI, J-XII, J-XIII, J-XIV, J-XV, J-XVI, J-XVII,J-XVIII, K-I, K-II, K-III, K-IV, K-V, K-VI, K-VII, K-VIII, K-IX, K-X,K-XI, K-XII, K-XIII, K-XIV, K-XV, K-XVI, K-XVII, K-XVIII, L-I, L-II,L-III, L-IV, L-V, L-VI, L-VII, L-VIII, L-IX, L-X, L-XI, L-XII, L-XIII,L-XIV, L-XV, L-XVI, L-XVII, L-XVIII, M-I, M-II, M-III, M-IV, M-V, M-VI,M-VII, M-VIII, M-IX, M-X, M-XI, M-XII, M-XIII, M-XIV, M-XV, M-XVI,M-XVII, M-XVIII, N-I, N-II, N-III, N-IV, N-V, N-VI, N-VII, N-VIII, N-IX,N-X, N-XI, N-XII, N-XIII, N-XIV, N-XV, N-XVI, N-XVII, N-XVIII, O-I,O-II, O-III, O-IV, O-V, O-VI, O-VII, O-VIII, O-IX, O-X, O-XI, O-XII,O-XIII, O-XIV, O-XV, O-XVI, O-XVII, O-XVIII, P-I, P-II, P-III, P-IV,P-V, P-VI, P-VII, P-VIII, P-IX, P-X, P-XI, P-XII, P-XIII, P-XIV, P-XV,P-XVI, P-XVII, P-XVIII, Q-I, Q-II, Q-IV, Q-V, Q-VI, Q-VII, Q-VIII, Q-IX,Q-X, Q-XI, Q-XII, Q-XIII, Q-XIV, Q-XV, Q-XVI, Q-XVII, Q-XVIII, R-I,R-II, R-III, R-IV, R-V, R-VI, R-VII, R-VIII, R-IX, R-X, R-XI, R-XII,R-XIII, R-XIV, R-XV, R-XVI, R-XVII, R-XVIII, preferably the combinationsare as follows A-I, G-VII, L-XII.

wherein each heavy chain CDR3 sequence comprises one or two amino acidsubstitutions, deletions and/or insertions, preferably one or two aminoacid substitutions as listed above in Table 1; wherein each heavy chainCDR2 sequence comprises one or two amino acid substitutions, deletionsand/or insertions, preferably one or two amino acid substitutions aslisted above in Table 1; wherein each heavy chain CDR1 sequencecomprises one, two or three amino acid substitutions, deletions and/orinsertions, preferably one, two or three amino acid substitutions aslisted above in Table 1; wherein each light chain CDR3 sequencecomprises one or two amino acid substitutions, deletions and/orinsertions, preferably one or two amino acid substitutions as listedabove in Table 2; wherein each light chain CDR2 sequence comprises oneor two amino acid substitutions, deletions and/or insertions, preferablyone or two amino acid substitutions as listed above in Table 2; whereineach light chain CDR1 sequence comprises one, two or three amino acidsubstitutions, deletions and/or insertions, preferably one, two or threeamino acid substitutions as listed above in Table 2.

In preferred embodiments of the fourth aspect, the antibody orantigen-binding fragment thereof comprising one or more CDRs, a set ofCDRs or a combination of sets of CDRs as described herein comprises saidCDRs in a human antibody framework.

In a preferred embodiment of the fourth aspect the antibody is apolyclonal or monoclonal antibody.

In a further preferred embodiment of fourth aspect the antibody is ahuman or humanized antibody.

In a further embodiment of the fourth aspect the present invention isdirected to an isolated monoclonal antibody, wherein the antibodybinding portion comprises a light chain comprising an amino acidsequence at least 80%, preferably at least 85%, preferably at least 90%,more preferably at least 91%, more preferably at least 92%, morepreferably at least 93%, more preferably at least 94%, more preferablyat least 95%, even more preferably at least 96%, even more preferably atleast 97%, even more preferably at least 98%, even more preferably atleast 99% or most preferably at least 100% identical to the light chainvariable region amino acid sequence set forth in SEQ ID NO: 7, SEQ IDNO: 11, or SEQ ID NO: 13.

In a further preferred embodiment the antibody or antibody bindingportion comprises a heavy chain comprising an amino acid sequence atleast 80%, preferably at least 85%, preferably at least 90%, morepreferably at least 91%, more preferably at least 92%, more preferablyat least 93%, more preferably at least 94%, more preferably at least95%, even more preferably at least 96%, even more preferably at least97%, even more preferably at least 98%, even more preferably at least99% or most preferably at least 100% identical to the heavy chainvariable region amino acid sequence set forth in SEQ ID NO: 8, SEQ IDNO: 12, or SEQ ID NO: 14.

In a fifth aspect, the present invention is further directed to apharmaceutical composition, which comprises a nucleic acid of theinvention, an expression vector or the invention or an antibody of theinvention and a pharmaceutically acceptable carrier or excipient.

As used herein, the expressions “is for administration” and “is to beadministered” have the same meaning as “is prepared to be administered”.In other words, the statement that an active compound “is foradministration” has to be understood in that said active compound hasbeen formulated and made up into doses so that said active compound isin a state capable of exerting its therapeutic activity.

The terms “therapeutically effective amount” or “therapeutic amount” areintended to mean that amount of a drug or pharmaceutical agent that willelicit the biological or medical response of a tissue, a system, animalor human that is being sought by a researcher, veterinarian, medicaldoctor or other clinician. The term “prophylactically effective amount”is intended to mean that amount of a pharmaceutical drug that willprevent or reduce the risk of occurrence of the biological or medicalevent that is sought to be prevented in a tissue, a system, animal orhuman by a researcher, veterinarian, medical doctor or other clinician.

Various delivery systems are known and can be used to administer thepharmaceutical composition of the invention, e.g., encapsulation inliposomes, microparticles, microcapsules, recombinant cells capable ofexpressing the mutant viruses, receptor mediated endocytosis (see, e.g.,Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introductioninclude, but are not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The composition may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Administration can be systemic or local.

The pharmaceutical composition can be also delivered in a vesicle, inparticular a liposome (see Langer (1990) Science 249:1527-1533; Treat etal. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer,Lopez Berestein and Fidler (eds.), Liss, New York, pp. 353-365;Lopez-Berestein, ibid., pp. 317-327; see generally ibid.).

In certain situations, the pharmaceutical composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201).In another embodiment, polymeric materials can be used; see, MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974). In yet another embodiment, a controlled releasesystem can be placed in proximity of the composition's target, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138,1984).

The injectable preparations may include dosage forms for intravenous,subcutaneous, intracutaneous and intramuscular injections, dripinfusions, etc. These injectable preparations may be prepared by methodspublicly known. For example, the injectable preparations may beprepared, e.g., by dissolving, suspending or emulsifying the antibody orits salt described above in a sterile aqueous medium or an oily mediumconventionally used for injections. As the aqueous medium forinjections, there are, for example, physiological saline, an isotonicsolution containing glucose and other auxiliary agents, etc., which maybe used in combination with an appropriate solubilizing agent such as analcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol,polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80,HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)],etc. As the oily medium, there are employed, e.g., sesame oil, soybeanoil, etc., which may be used in combination with a solubilizing agentsuch as benzyl benzoate, benzyl alcohol, etc. The injection thusprepared is preferably filled in an appropriate ampoule. Apharmaceutical composition of the present invention can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

The term “acceptable carrier or excipient” as used herein, refers to adiluent, adjuvant, excipient, or vehicle with which the therapeuticagent is administered. Such pharmaceutical carriers can be sterileliquids, such as saline solutions in water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. A saline solution isa preferred carrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsions,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. The compounds ofthe invention can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include those formed with free aminogroups such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with free carboxyl groupssuch as those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc. Examples of suitable pharmaceutical carriersare described in “Remington's Pharmaceutical Sciences” by E. W. Martin.Such compositions will contain a therapeutically effective amount of thecompound, preferably in purified form, together with a suitable amountof carrier so as to provide the form for proper

In a preferred embodiment of pharmaceutical further comprises ananti-neoplastic agent as outlined above.

In a preferred embodiment of the pharmaceutical composition furthercomprises an antibody against oncogenic or stromal proteins. Suchantibodies are known in the art.

In a sixth aspect the present invention is directed to a kit, whichcomprises an isolated nucleic acid fragment or a vector in a containerand an instruction for using the isolated nucleic acid fragment inpreventing, treating or delaying a neoplasm.

The present invention among other things also relates to:

-   1. A nucleic acid comprising a nucleotide sequence encoding a mammal    ErbB3 H584F mutant protein said protein comprising a sequence of    amino acid as set forth in SEQ ID NO: 2 or a functional fragment    thereof, wherein one or more of the nucleotide codons encoding the    protein that occur with low frequency in proteins expressed in    mammal cells have been replaced with nucleotide codons present on    nucleic acids encoding highly expressed proteins.-   2. An expression vector comprising the nucleic acid of item 1    operatively linked to a promoter or to a regulatory transcriptional    element, belonging preferentially but not exclusively to the    following classes: bacterial plasmid, adenovirus, poxvirus,    vaccinia, fowlpox, herpes, adeno-associated virus (AAV), alphavirus,    lentivirus, lambda phage, lymphocytic choriomeningitis virus,    Listeria sp, Salmonella sp.-   3. A method for preventing, treating or delaying neoplams in a    mammal, which method comprises administering to a mammal, to which    such preventiom treatment or delay is needed or desirable a nucleic    acid of item 1 encoding Erbb3 H584F mutant protein, or a functional    fragment thereof, whereby an immune response is generated against    said neoplasm and said neoplasm is prevented, treated or delayed.-   4. The method of item 3, wherein the mammal is a human, mouse, rat    or dog.-   5. The method of item 3, wherein the nucleic acid encoding Erbb3    H584F and its extracellular domain comprises a nucleic acid set    forth in SEQ ID NO: 1 and SEQ ID NO: 3.-   6. The method of item 3, further comprising administering the    nucleic acid by DNA electroporation in the muscle or to the neoplasm    in situ.-   7. The method of item 3, further comprising administering an immune    response potentiator to the mammal.-   8. The method of item 3, wherein the nucleic acid is co-administered    with a pharmaceutically acceptable carrier or excipient.-   9. The method of item 3, wherein the nucleic acid is co-administered    in combination with a known therapeuticanti-neoplasm regimen.-   10. The method of item 9, wherein the anti-neoplasm agent is    selected from the group consisting of an anti-angiogenic agent, an    alkylating agent, an antimetabolite, a natural product, a platinum    coordination complex, an anthracenedione, a substituted urea, a    methylhydrazine derivative, an adrenocortical suppressant, a    hormone, an antagonist, an oncogene inhibitor, a tumor suppressor    gene or protein, a therapeutic antibody and an anti-oncogene    oligonucleotide.-   11. The method of item 3, wherein the neoplasm to be prevented    treated or delayed is selected from the group consisting of adrenal    gland, anus, auditory nerve, bile ducts, bladder, bone, brain,    breast, central nervous system, cervix, colon, ear, endometrium,    esophagus, eye, eyelids, fallopian tube, gastrointestinal tract,    head and neck, heart, kidney, larynx, liver, lung, mandible,    mandibular condyle, maxilla, mouth, nasopharinx, nose, oral cavity,    ovary, pancreas, parotid gland, penis, pinna, pituitary, prostate    gland, rectum, retina, salivary glands, skin, small intestine,    spinal cord, stomach, testes, thyroid, tonsil, urethra, uterus,    vagina, vestibulocochlear nerve and vulva neoplasm.-   12. An antibody generated with the method of item 3, which antibody    binds to an epitope in an extracellular domain of the ErbB3 protein,    or a functional fragment thereof and exhibits one or more of the    following properties:    -   i. Inhibition of heregulin, epiregulin, betacellulin, epigen or        biregulin-mediated signalling through ErbB3;    -   ii. Inhibition of proliferation of cells expressing ErbB3;    -   iii. The ability to decrease levels of ErbB3 on cell surfaces;    -   iv. Inhibition of VEGF secretion of cells expressing ErbB3;    -   v. Inhibition of the migration of cells expressing ErbB3; and    -   vi. Inhibition of spheroid growth of cells expressing ErbB3.-   13. The antibody of item 12, which is a polyclonal or monoclonal    antibody.-   14. The antibody of item 12, which is a human or humanized antibody.-   15. An isolated monoclonal antibody of item 14, wherein the antibody    binding portion comprises a light chain comprising an amino acid    sequence at least 80% identical to the light chain variable region    amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID    NO: 13.-   16. An isolated monoclonal antibody of item 12, wherein the antibody    binding portion comprises a heavy chain comprising an amino acid    sequence at least 80% identical to the heavy chain variable region    amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID    NO: 14.-   17. A pharmaceutical composition, which pharmaceutical composition    comprises an antibody of item 12 and a pharmaceutically acceptable    carrier or excipient.-   18. The pharmaceutical composition of item 17, which further    comprises an antineoplasm agent.-   19. The pharmaceutical composition of item 17, which further    comprises an antibody against oncogenic or stromal proteins.-   20. A kit, which kit comprises an isolated nucleic acid fragment of    item 1 in a container and an instruction for using the isolated    nucleic acid fragment in preventing, treating or delaying a    neoplasm.

FIGURE LEGENDS

FIG. 1: Panel A depicts the structure of the plasmidpTK1-A-HER3-H584F-FL_(opt); which encodes a codon optimized version offull length human HER3 carrying a mutation at position 584 from His toPhe. Panel B depicts the structure of the plasmidpTK1-A-HER3-H584F-ECD_(opt), which encodes a codon optimizedC-terminally truncated form of human HER3 comprising the extracellulardomain. Panel C depicts the results of an ELISA assay of sera of BALB/cmice (8 animals) immunized with the vaccine. A significant reactivityagainst recombinant HER3 was observed for all sera.

FIG. 2: Panel A depicts a Western blot of cell extracts of variouscancerous cell lines with an anti-HER3 antibody. It is evident that theOvCar 3 and OvCar8 cell lines have the highest levels of HER3expression. Panel B depicts a Western blot of cell extracts of OvCar 3and OvCar8, wherein phosphorylated HER3 is detected by an anti-pHER3-Tyr1298 phosphorylation specific antibody in the presence of differentamounts of HER3-specific antibodies A3 and A4 of the invention.

FIG. 3: Panel A depicts a Western blot of cell extracts of OvCar 3 andOvCar8, wherein phosphorylated AKT is detected by an anti-pAKT Ser 473phosphorylation specific antibody in the presence of different amountsof HER3-specific antibodies A3 and A4 of the invention. Panel B depictsa Western blot of OvCar 3 and OvCar8 cell lines with a HER3 specificantibody in the presence of different amounts of HER3-specificantibodies A3 and A4 of the invention.

FIG. 4: Panel A depicts a Western blot of cell extracts of HeLa cells,adherently growing primary cell cultures established from MalignantPleural Effusions (MPEs) and spherically growing MPEs with fivedifferent antibodies directed against EGFR, ErbB2, HRG, Tubulin andErbB3/Her2. Panel B depicts the inhibition of phosphor-HER3 expressionin the control (UT), by an unspecific IgG (Contr. IgG), with the HER3specific antibodies of the invention A3 and A4 (A3 and A4,respectively).

FIG. 5: Panel A depicts the results of an assay evaluating tumor cellproliferation inhibition by antibodies A3 and A4. Panel B depicts theresults of an assay evaluating inhibition of spherer formation of MPEsby antibodies of the invention A3 and A4.

FIG. 6: Panel A depicts the results of an experiment, wherein theinhibitory activity of HER3 specific A3 and A4 antibodies of theinvention on tumors is evaluated that are formed by implantation ofcells from the human tumor cell line BxPC3 in CD1 nude mice. Panel Bdepicts the results of an experiment, wherein the efficacy of anti-HER3antibodies of the invention A3 and A4 in the BALB/neuT model isevaluated.

FIG. 7: Panel A depicts the results of an experiment evaluating theeffect of A3 and A4 on tumor multiplicity in the BALB/neuT model overtime. Panel B depicts the results of an experiment evaluating theinfluence of 10% serum, no serum, the presence of NRG alone or ofantibodies A3 or A4 in each case with or without Her2 specific antibodyHerceptin™:

FIG. 8: Panel A depicts the results of the epitope mapping assay of A3antibody. Wells containing the epitope QCNGHCFGPNPNQCC and positivecontrols with human ErbB3 recombinant protein are shown by arrows. PanelB depicts the position of the epitope (in yellow) within the crystalstructure of ErbB3. Domains I, II, III and IV are indicated withdifferent color codes.

FIG. 9: Panel A depicts a quantification of humanized A4, A4 and ControlIgG immunoglobulins in a Coomassie stained SDS gels run under denaturingor non-denaturing conditions. Panel B depicts the results of anexperiment comparing the specificity of binding of humanized A4 (humA4)and murine A4 (A4) to human HER3. Control IgG is an unspecific IgGcontrol.

FIG. 10: Depicts the amino acid sequences of the Variable heavy andlight chain domains of A3, A4 and humanized A4. In each case the aminoacids constituting the complementarity determining region (CDR) 1, 2 or3 are indicated by bold print.

EXAMPLES Example 1 A Genetic Vaccine Against a Genetic Variant of HER3

In order to obtain a strong immune response against HER3 and inparticular in order to induce neutralizing antagonistic antibodiesanti-HER3 in the organism, a genetic vaccination approach has beenadopted, which is based on DNA electroporation in skeletal muscles(DNA-EP, ref 21). This technology allows both the use of appropriatelyengineered modified variants of the antigen of interest, and allows itsendogenous expression in the muscle and in antigen presenting cells. Aplasmid vector has been utilized which carries a modified HER3 cDNAwhose codons have been optimized for their presence in the coding regionof proteins highly expressed in human cells. This modified cDNA alsoexpresses a mutant form of the receptor with a single amino acidsubstitution H584F described in SEQ ID:1. The amino acid sequence of theprotein is depicted in SEQ ID:2. In the absence of the ligand, HER3 ispresent on the cell surface in a closed conformation which is tetheredtogether by intramolecular bonds. When bound to its ligand, HER3 adoptsan extended conformation which exposes receptor domains responsible forthe heterodimerization with other ErbB receptors and other RTK partners.Since mutant H584F is constantly held in an open conformation [22], alsoin the absence of the ligand, we postulated that immunizations with thisvariant should increase the probability of obtaining neutralizingantibodies.

The immunization protocol consisted of 2 injections in the quadricepsmuscle with 50 μg of plasmid pTK1-A-HER3-H584F-FL_(opt) (FIG. 1A)followed by 2 injections with 50 μg of plasmidpTK1-A-HER3-H584F-ECD_(opt) (FIG. 1B), carrying the cDNA coding for theECD domain with the mutation H584F (nucleotide sequence in SEQ ID:3,amino acid sequence in SEQ ID:4). DNA injections were spaced 2 weeksfrom one another. DNA was formulated in Phosphate Buffered Saline (PBS)at a concentration of 1 mg/ml. Antigen expression was driven bycytomegalovirus promoter (CMV) upstream of the intron A and the HER3cDNA was followed by the bovine Growth Hormone (bGH) polyadenylationsignal. DNA electroporation in vivo was carried out with a BTXelectroporator, model BTX 830, and plate electrodes at a distance of 0.5cm (BTX, Harward apparatus) using the following electrical conditions inLow voltage: 2 pulses of 60 msec at 100V, with 250 msec pause betweenpulses. All BALB/c immunized mice (8 animals) responded to the vaccineand their sera showed a significant reactivity against recombinant HER3in an ELISA assay (FIG. 1C).

Two weeks after the fourth and last immunization, mice were euthanizedand spleens and lymphnodes removed. After a standard fusion protocolwith murine myeloma cells as described in Harlow et al, “Antibodies: alaboratory manual”, more than 100 hybridoma clones were isolated bylimiting dilution and their supernatant tested again for binding torecombinant HER3 in vitro.

Example 2 Biochemical and Functional Analysis

Among the hybridomas described above, two antibodies, A3 and A4, werefurther selected for a biochemical and functional characterization. Inparticular, the following assays have been performed:

-   -   Bioacore analysis. Affinity for HER3 and k_(on)/k_(off) were        determined by Surface Plasmon Resonance using a Biacore T100        instrument. A biosensor chip CM5 (GE Healthcare) was covalently        bound to an anti-Fc (8000 RU) and then treated with antibodies        at different concentrations (<1000 RU). Recombinant protein        HER3-ECD-Fc (RnD Systems) in HBS-EP buffer at the analyte        concentration 1:1 was injected for 2 minutes at 30 μl/min,        followed by a 10 minutes dissociation phase.    -   Cross-reactivity with mouse HER3 receptor and with other human        ErbB receptors. In order to assess potential cross-reactivity        with other ErbB receptors, cells negative for ErbB receptors        expression (CHO) were transiently transfected with expression        vectors (pCDNA3) for human or mouse HER3, HER1/EGFR, HER2 or        HER4. 5×10⁵ cells were transfected in 6 cm plates with 5 μg of        plasmid DNA using Lipofectamine 2000 (Gibco). After 48 hours,        cells were trypsinized, washed with culture medium and incubated        with 10 μg/ml of each antibody in FACS buffer (PBS, 1% FBS).        After a wash in FACS buffer, an anti-mouse IgG conjugated with        Phycoerythrin (PE, Becton Dickinson) was added and cells were        analyzed by a FACSCalibur (Becton Dickinson).    -   Ligand blocking assay. In order to measure the ability of        antibodies to inhibit the binding of neuregulin (NRG) to HER3,        competition assays with the ligand were carried out using an        ELISA assay. In detail, the recombinant protein HER3-ECD-Fc was        bound to Nunc Maxisorp plastic plates over night at the        concentration of 1 μg/ml in PBS at 4° C. Anti-HER3 antibodies        were then added for 1 hour at 37° C., followed by an incubation        for 1 hour with 90 ng/100 μl/well with NRG-1β (RnD Systems,        final concentration 10 nM). After 2 washes with PBS, 0.05%        Tween20, the signal was measured by incubation with a        biotinylated goat anti-NRG-1β antibody (RnD Systems) and        Streptavidin conjugated with peroxidase and utilizing an HRP        substrate (Pierce). Data were acquired with an ELISA reader at        450 nm.    -   HER3 and Akt Activation/Phosphorylation assays. ELISA assays        have been performed to check whether anti-HER3 antibodies were        able to block NRG-mediated HER3 activation. MCF-7 cells, were        grown to 70% confluence in 6 cm plates and then starved 0/N in        culture medium (RPMI) with 0.1% BSA. Cells were then incubated        for 1 hour with anti-HER3 antibodies and then treated with 3.3        nM (100 ng/ml) NRG1β in medium comprising 0.1% BSA for 20 min at        37° C. Cell extracts were prepared and ELISA assays performed        according to the instructions of the kit DUOset IC Human        Phospho-ErbB3 and Human Phospho-Akt (RnD Systems). Percent        inhibition was calculated using as reference an unrelated IgG as        negative control.    -   Receptor Internalization/Degradation assays. Disappearance of        the HER3 receptor from cell surface following its        internalization and/or degradation interferes with intracellular        signalling, and therefore with cell transformation and/or        maintenance of tumorigenicity. These assays were carried out        using FACS analysis. In particular, MCF-7 cells were        trypsinized, resuspended in FACS buffer and counted. 1×10⁶ cells        in each tube were incubated with antibodies for 3 hours at        37° C. using a control isotype antibody. After a wash in FACS        buffer, a further incubation with the same antibodies was        executed for 30 min at 4° C., followed by an incubation with an        anti-mouse IgG-PE using the same conditions. Cells were then        fixed in PBS, with 10% formaldehyde and analyzed by FACSCalibur.        Percent internalization was calculated using a reference IgG as        negative control.    -   Proliferation assays. In order to assess the ability of        anti-HER3 antibodies to inhibit cell proliferation induced by        NRG, the following experiments were performed in vitro using        MCF-7 cells. 2000 cells/well were seeded in 96 well plates in        culture medium conditioned with FBS over night. Following that,        cells were incubated in quadruplicates with the anti-HER3        antibodies at a concentration of 100 μg/ml in medium conditioned        with 0.5% FBS for 1 hour at 37° C., and then stimulated with 30        ng/ml of NRG1β for 72 hours. The Alamar Blue (Biosource) dye was        then added, and after incubation for 90 minutes, absorbance was        measured with an ELISA reader at 590 nm. Percent inhibition was        calculated using a reference IgG as negative control.        The following table summarizes the results obtained and the        features of A3 and A4 monoclonal antibodies.

NRG Inhibition of Inhibition or Binding to Ligand pHER3 binding pAktcell Specificity murine Biacore Blocking stimulation Inhibition,Receptor proliferation, Ab (other HER) HER3 (Kd, nM) (nM) nM/Max % % at100 nM Internalization, % MCF-7, % A3 Only Yes 7.4 ± 0.3 1.1 ± 0.45.0/80 79 ± 9.5 48 ± 3 60 ± 15 HER3 A4 Only No 2.4 ± 0.1 2.8 ± 0.47.0/78 69 ± 6.2 43 ± 5 92 ± 12 HER3

Example 3 Efficacy of Anti-HER3 Antibodies on Human Ovarian Cancer CellLines

HER3 expression has been reported in ovarian carcinoma [23]; hencecancer cells lines of this origin can be utilized to test the antitumoreffect of anti-HER3 antibodies. In order to select the most suitablecell lines, a Western Blot was performed with a commercial anti-HER3antibody (1B2E, Cell Signalling), using cell extracts derived fromvarious cell lines: OvCar429, OvCar3, OvCar4, OvCar8, Skov3 and Igrov.50 μg of each cell extract were loaded on a NuPage gel 4-12%(Invitrogen) and blotted onto nitrocellulose. After blocking for 30minutes with PBS, 5% milk, 0.5% Tween20, the anti-HER3 antibody wasadded and incubated over night at 4° C. After several washes, the filterwas incubated with a secondary anti-rabbit IgG conjugated withperoxidase for one hour at room temperature, washed and treated with anECL substrate (Amersham). Results, in FIG. 2A, show that the OvCar 3 andOvCar8 cell lines have the highest levels of HER3 expression.

In order to verify the effect of A3 and A4 antibodies on signaltransduction in OvCar3 and OvCar8, cells were seeded in 6 cm plates,grown to 70% confluence and starved for 20 hours in RPMI with theaddition of 0.05% BSA. Antibodies A3 or A4 were added at theconcentrations of 250 nM, 83 nM and 28 nM for 90 minutes. Then cellswere stimulated with NRG at the concentration of 80 ng/ml for 20 minutesand protein extracts quickly prepared. A commercial anti-pHER3 and ananti-pAKT antibody (Cell Signalling) were used to study the effect onsignalling transduction. FIGS. 2B and 3A, respectively, show that theinhibition of pHER3 and pAKT is dose-dependent on both cell lines. Inorder to verify the effect of A3 and A4 monoclonal antibodies on totalHER3 in both cell, the same commercial anti-HER3 antibody describedbefore has been utilized in Western Blots. The results show that both A3and A4 induce a strong degradation of the receptor in these cell lines(FIG. 3B).

Example 4

Expression of ErbB Receptor Members in Spheroids from Primary LungCancer Cells.

It has been recently shown that Malignant Pleural Effusions (MPEs) areone of the best sources of primary lung tumor cells, because that caneasily be propagated in vitro and in vivo and reproduce the naturalheterogeneity of tumors [24]. The preparation of primary cultures fromMPEs has been performed from effusions obtained from patients with lungadenocarcinomas. In order to collect cells, the pleural fluid has beencentrifuged for 10 min at 1500 rpm using a centrifuge Heraeus SepatechOmnifuge 2.0 RS. The cell pellet has been washed once in PBS andresuspended in 1% BSA/2 mM EDTA/PBS. The cell suspension was stratifiedon a Histopaque solution (Sigma-Aldrich) whose density was adjusted to1,065 g/ml by addition of PBS and the gradient thus formed has beencentrifuged at 800 g for 20 minutes at room temperature. The upperphase, containing a small amount of lymphocytes and abundant tumor cellshas been collected at the interphase (about 35 ml) and after a wash in1% BSA/2 nM EDTA/PBS, cells have been resuspended in spheroid culturemedium (see below). The gradient pellet, composed by erythrocytes,lymphocytes and a smaller number of tumor cells, has been resuspendedand incubated with ACK buffer in order to lyse erythrocytes for 15 minat room temperature. After the lysis, cells were centrifuged again andwashed with PBS. Afterwards, cells were plated in culture medium foradherence (see below)—

Adherent Culture Conditions:

RPMI medium/GlutaMax with 10% FBS and 1 mM Pen/Strep. In theseconditions, primary tumor cells form a monolayer on plastic cultureplates.

Spheroid Culture Conditions:

Cells have been resuspended at a density of 100,000/ml in Dulbecco'smodified Eagle's medium/F12 (Invitrogen) supplemented with 1% BSA, 0.5%Glucose, 20 μg/ml insulin (Sigma-Aldrich), 15 mM Hepes, B27 withoutretinoic acid (Invitrogen), 4 μg/ml heparin (Sigma-Aldrich), 20 ng/mlepidermal growth factor (EGF) and 20 ng/ml basic fibroblast growthfactor (bFGF) and plated in non-treated plates for cell culture (Falcon)or in Ultra-Low binding plates (Corning). Under these conditions, cancercells with stem cell features can form three-dimensional structurescalled spheroids. Growth factors (EGF and BFGF) have been added every2-3 days and culture medium replaced every 7 days. After theirformation, spheres were disaggregated mechanically or via incubation inAccumax (Innovative Cell Technologies Inc.). The cell suspension hasbeen plated again in the same conditions described above in order toshow spheroid propagation (sphere formation assay).

Protein extracts have been prepared from cells derived from MPEs andcultured in adherence or as spheroids, and have been analyzed by WesternBlotting. In detail, 25 μg of extracts have been loaded onto NuPage gels4-12% (Invitrogen) and transferred on nitrocellulose membranes. In orderto detect HER3 expression, the anti-HER3 antibody from Santa Cruz hasbeen utilized. Furthermore, also EGFR and HER2 expression has beenmeasured by Western Blotting with anti-EGFR (Santa Cruz) and anti-HER2antibodies (RnD systems) respectively. As negative control, extractsfrom the cervical cancer cell line HeLa have been utilized. HER3expression is approximately 5-fold higher when cells are grown asspheroids as compared to adherent cells (FIG. 4A). Likewise, also theexpression of HER3 ligand, heregulin α, β (HRG-α, β measured withanti-HRG, NeoMarkers) increases in spheroids. In contrast, EGFR and HER2expression is completely shut off in spheroid cultures. These datasuggest that HER3 and the PI3K/AKT signalling axis may have an importantrole for the cancer stem cell propagation in lung cancer.

Example 5 Inhibition of Signalling, Proliferation and Spheroid Formationof Primary Lung Cancer Cells.

In order to assess the effect of anti-HER3 antibodies on MPE-derivedcells, primary cultures have been plated in spheroid medium in theabsence of FBS and treated with antibodies A3 or A4 at the concentrationof 10 μg/ml for 1 hour before the preparation of protein extracts. Theeffect on HER3 phosphorylation has been monitored by Western Blottingusing an anti-pHER3 antibody (Cell Signaling). As shown in FIG. 4B,antibodies A3 and A4 were able to reduce 23 and 67%, respectively, thelevel of HER3 phosphorylation. An antibody of the same isotype used asnegative control did not show any effect.

In order to verify the effect of anti HER3 antibodies on the ability ofcells to grow in adherent conditions, cells derived from MPEs have beencultured with A3 or A4 at the concentration of 10 μg/ml, 2 wells foreach experimental point. After 10 days cells were detached, stained withTrypan Blue (Sigma), and the number of living cells determined at aZeiss Axjovert 25 microscope (Jena, Germany). Results are shown in FIG.5A and indicate that tumor cell proliferation is inhibited withAntibodies A3 and A4, 22 and 36% respectively.

Lastly, the inhibitory activity of A3 and A4 on spheroid formation, hasbeen assessed in a spheroid forming assay. After trypsin treatment,MPE-derived cells from adherent cultures have been seeded in 24-wellplates in spheroid forming condition in the presence of A3, A4 orisotype negative control, at the concentration of 10 μg/ml, 3 wells foreach experimental condition, and kept in culture for 10 days. At the endof this incubation time, sphere number has been determined with a ZeissAxjovert 25 microscope (Jena, Germany). The total number of spheres/wellwas determined and the average number for the 3 wells was calculated.Sphere counts has been determined by two independent operators withcomparable results. FIG. 5B shows that both A3 and A4 are able tosignificantly reduce the number of spheres by 22% and 46% respectively.

Example 6 Antitumor Efficacy on Pancreatic Tumors in Nude Mice

In order to assess the therapeutic potential of anti-HER3 antibodies, A3and A4 have been tested in CD1 nude mice bearing palpable tumors derivedfrom the implantation of cells from the human tumor cell line BxPC3. Indetail, 1×10′ BxPC3 cells have been injected subcutaneously (s.c.) inthe right flank of each mouse in the presence of matrigel (Reducedgrowth factor matrigel, BD bioscience). After 7 days, when tumors hadreached the size of 200 mm³, mice were randomized in homogeneous groupsand treated with 3 weekly injections of A3, A4 or control antibody ofthe same isotype at the dose of 25 mg/kg i.p. Results are presented inFIG. 6A and show that both A3 and A4 are able to block pancreatic tumorprogression.

Example 7 Antitumor Efficacy on Spontaneous Mammary Tumors in TransgenicMice.

In a vast majority of cases the efficacy of therapeutic compoundsagainst cancer is studied in immuno-deficient mice implanted with s.ctumors, as described in the previous example. However these models,while allowing to show efficacy on tumor cells of human origin, presentthe limitation of not being able to evaluate the therapeutic effect onspontaneous tumorigenesis, and to appreciate the influence of the immunesystem on the system of interest.

For this reasons, the BALB/neuT mouse model has been utilized. Theseanimals express the oncogenic form of the rat HER-2/neu receptor,specifically in the mammary gland. This tissue specific expressioninduces the appearance of hyperplasia at the 10^(th) week of life and ofadenocarcinomas between the 15^(th) and the 20^(th) week of life [25].Evidences in literature suggest that in HER-2/neu transgenic mice, HER-3is expressed and has an active role in the development andaggressiveness of mammary tumors [26]. Furthermore, vaccination studiesperformed with whole cell vaccines expressing ErbB family receptormembers, including also HER3, have shown some antitumor efficacy in thismurine model [27].

In order to assess the therapeutic efficacy of anti-HER3 antibodies inthe BALB/neuT model, 2 groups of 4 mice have been utilized in which thesize of a reference developing tumor was about 40 mm³ as measured byultrasound with the instrument VEVO 770 (Visualsonics), and bearing lessthan 2 tumors/mouse. The first group has been treated with a negativecontrol antibody of the same isotype, while the second has been treatedwith the antibody A3. Antibody A4 has not been utilized because it doesnot cross-react with mouse HER-3. Antibodies were injected twice/weeki.p. at the dose of 25 mg/kg. Every 7 days the size of reference tumorswas measured by ultrasound imaging and the tumor multiplicity bypalpation. FIG. 6B shows that antibody A3, compared with the negativecontrol, is able to slow down the growth of the reference tumors.Furthermore, FIG. 7A shows a significant effect on tumor multiplicityover time (p<0.01). As a final confirmation of these results, at the endof the study (week 4), mice have been euthanized, tumors explanted andweight determined.

The table below shows the difference between the experimental groups.

A3 Contr. IgG #1 0.869 5.934 #2 0.695 3.151 #3 3.282 7.500 #4 1.1216.389 Media 1.492 5.744 St-Dev 1.2 1.8

Example 8

Synergic Efficacy of A3 and A4 Monoclonal Antibodies with Herceptin™.

In literature there are evidences that the combination of monoclonalantibodies against EGFR and HER2 can lead to increased anti-tumorefficacy as a consequence of the simultaneous inhibition ofheterodimeric receptor complexes that contribute to cell transformation[28]. In order to assess whether A3 and A4 antibodies could have asynergistic effect with the anti-HER2 monoclonal antibody Herceptin™, acombination study has been performed on MCF-7 cells. 2000 cells/wellhave been seeded in 96 well plates in culture medium with 10% FBS overnight. Cells have then been washed with PBS and incubated inquadruplicates with the antibodies at the concentration of 10 mg/ml, assingle agent or in combination with Herceptin™ at the sameconcentration, in medium with 1% FBS and incubated for 6 days. The MTTdye (Sigma) has been added and incubated for 60 minutes. Absorbance hasthen been measured with an ELISA reader at 570 nm. The results in FIG.7B show that MCF-7 proliferation at low serum concentrations and in thepresence of NRG is not affected by Herceptin™. In contrast, both A3 andA4 inhibit cell proliferation approximately 30% and 40%, respectively.Inhibition increased above 50% when Herceptin™ is added to A3 or to A4.

Example 9 Sequence of CDRs.

In order to generate an antibody suitable for therapeutic application inhumans, the amino acid sequence of antibodies A3 and A4 has beendetermined by sequencing the cDNA obtained from the relevant hybridomas.Total RNA has been extracted from 10⁷ pelleted A3 and A4 hybridomacells, utilizing the Qiagen RNeasy mini kit (Cat No: 74104). Thepurified RNA has been resuspended in 50 μl of water and its qualitychecked by electrophoresis on a 1.2% agarose gel. cDNAs for V_(H) andV_(L) regions have been generated with primers specific for IgGs andconstant regions. cDNAs have been then amplified by PCR utilizingoligonucleotides that paired in the signal peptide sequence. AmplifiedDNA has been purified from gel and cloned in the vector pGEM-T-Easy(Promega). DNA extracted from clones V_(H) and V_(L) thus obtained hasbeen sequenced on both strands. The localization of the complementaritydetermining regions (CDRs) within the sequence has been determined byalignment with sequences of other antibodies [29]

Antibody A3

The DNA sequence of regions VL and VH is indicated in SEQ ID: 5 and 6.The corresponding amino acid sequences are shown in SEQ ID: 7 and 8.Five independent clones provided identical sequences.

Antibody A4

The DNA sequence of regions VL and VH is indicated in SEQ ID: 9 and 10.The corresponding amino acid sequences are shown in SEQ ID: 11 and 12.Six independent clones provided identical sequences with the exceptionof a single amino acid substitution in one clone.

Example 10 Antibody A3 Epitope Mapping.

To identify the epitope recognized by A3 antibody, a human ErbB3 peptidecollection consisting of 158 15mer peptides overlapping by 11 residues(Jerini Peptide Technologies, Berlin, Germany) has been utilized. Eachpeptide was resuspended in DMSO at 40 mg/ml and diluted in sodiumcarbonate buffer (10 mM, pH 9.6) at 1 μg/ml in 96 well plates (NuncMaxisorp) at a volume of 100 μl/well and incubated over night at 4° C.Human ErbB3 ECD-Fc protein (RnD) and buffer coated wells were used aspositive and negative controls of the assay, respectively. The dayafter, plates were washed with 200 μl PBS 1× pH 7.4 and blocked with PBS5% BSA for 2 hr at room temperature. After three washes with PBS, 0.05%Tween20 (PBST), plates were incubated for 2 hours at room temperaturewith A3 antibody diluted at 1 μg/ml in PBST, 1% BSA for 2 hours at roomtemperature. Plates were then washed three times in PBST and thenincubated with 1/5000 diluted goat anti-mouse IgG HRP conjugated (Abcam)for one hour. After three washing in PBST, 100 μl/well 3, 3′, 5,5′-Tetramethylbenzidine (TMB) Liquid Substrate (Sigma) were added andthe reaction stopped 20 minutes later with 50 μl/well Stop Reagent forTMB Substrate (Sigma). OD₄₅₀ was determined at an ELISA reader (PerkinElmer).

The results of the assay are shown in FIG. 8A and clearly show that amain single epitope is recognized by A3. The peptide sequence of theepitope is as follows: QCNGHCFGPNPNQCC (SEQ ID: 68). Analysis of ErbB3crystal structure [30] by CnD4.3 software mapped the epitope mostly inErbB3 finger loop of domain II (see FIG. 8B). These data suggest that A3antibody, in addition to the above described properties, has thepotential to act as inhibitor of ErbB3 dimerization.

Example 11 Antibody A4 Humanization.

In order to generate a humanized antibody, the method ofsuper-humanization described in Hwang et al [31] has been chosen.According to the method, antibody A4 has been classified in this manner:light chain kappa, with combination of Canonical Structures (CS) 6-1-1:heavy chain with combination of CS 1-2-x. Assignment of the J segment isJK2 for the light chain kappa, JH4 for the heavy chain. Since residue 71is a valine, for the CDR-H2 a CS2 has been assigned. The human germlineVH1-2*02 has been chosen for the heavy chain because there are evidencesthat VH1-2*02 should have a combination of CS 1-2-x. Furthermore, CDRsof the sequence VH1-2*02 (CDR-H1 and CDR-H2) are those most similar tothe CDR sequences of the murine antibody. For the light chain the choicehas been the human germline IGKV3-20*01 (CSs 6-1-1).

From the comparison of the murine A4 CDRs and the selected humangermline CDRs, the sequences indicated in SEQ ID: 13 and 14 (Light andHeavy chain respectively) have been designed, and synthetic genesgenerated by assembly of synthetic oligonucleotides followed by PCRaccording to the method described in Stemmer et al [32]. The sequencesgenerated are indicated in SEQ ID: 15 and 16. Synthetic genes have thenbeen cloned by recombination, using the Gateway system (Invitrogen) inplasmids pBS-EF1α-HC1 and pBS-EF1α-LCK carrying the synthetic genscoding for the human IgG type 1 Heavy Chain and kappa Light chainrespectively. The vectors thus obtained have been called pHC1-humA4 andpLCK-hum A4, respectively.

Example 12 Expression and Specificity of Hum-A4 Antibody.

A total of 5 μg of plasmids PHC1-humA4 and PLCK-humA4 has beentransfected with Lipofectamine 2000 (Invitrogen) in 293-EBNA cells(Invitrogen), seeded in 6 cm plates at a molar ratio of 7:3. As controlsthe cDNAs from a control immunoglobulin against a different antigen andthat of the original murine A4 antibody have been cloned in pBS-EF1α.

After transfection, cells have been incubated for one week beforecollecting the supernatant. Immunoglobulins have been quantified with aForteBio biosensor (CA, US) and analyzed by Coomassie staining in nativeand denaturing conditions (FIG. 9A). All immunoglobulins were producedin similar amounts.

In order to assess the ability of IgGs to bind protein HER3, an ELISAassay has been performed utilizing amounts from 5 to 0.04 mg ofIgGs/well. Results in FIG. 9B show that humA4 is able to bind human HER3with an efficiency similar to the murine antibody.

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1. A method of preventing, treating, or delaying neoplasms in a mammal,which comprises administering to a subject a nucleic acid or anexpression vector comprising the nucleic acid, wherein the nucleic acidcomprises a nucleotide sequence encoding a polypeptide, wherein thepolypeptide comprises: (a) a mammalian ErbB3 mutant protein comprising,in comparison to the respective wild type ErbB3 protein, an amino acidsubstitution, which changes the conformation of the extracellular domain(ECD) of said ErbB3 to an extended conformation, (b) a N- and/orC-terminal deletion fragment of (a) comprising at least the ECD of theErbB3 protein, or (c) a variant of (a) or (b), which has at least 80%amino acid sequence identity to the amino acid of (a) or (b).
 2. Themethod of claim 1, wherein one or more nucleotide codons encoding (a),(b), or (c) that occur with low frequency in proteins expressed inmammalian cells is substituted with nucleotide codons that occur innucleic acids encoding highly expressed proteins.
 3. The method of claim2, wherein the substitution is at amino acid position 584 according toSEQ ID NO: 2 or at an amino acid that occupies an analogous position inan ErbB3 protein.
 4. The method of claim 3, wherein the amino acid atposition 584 according to SEQ ID NO: 2 or the an amino acid occupying ananalogous position is substituted by Phenylalanine (Phe).
 5. The methodof claim 1, wherein the polypeptide is selected from the groupconsisting of SEQ ID NO: 2 and SEQ ID NO:
 4. 6. The method of claim 1,wherein the nucleic acid in the expression vector is operatively linkedto a promoter or to a regulatory transcriptional element.
 7. The methodof claim 1, wherein the expression vector is selected from the groupconsisting of a bacterial plasmid, an adenovirus, a poxvirus, a vacciniavirus, a fowl pox virus, a herpes virus, an adena-associated virus(AAV), an alphavirus, a lentivirus, a lambda phage, a lymphocyticchoriomeningitis virus, a Listeria sp., and a Salmonella sp.
 8. Themethod of claim 1, wherein an immune response is generated against saidneoplasm.
 9. The method of claim 9, wherein the immune response is a Tand/or B cell response.
 10. The method of claim 1, wherein the mammal isa human, mouse, rat, or dog.
 11. The method of claim 1, wherein thenucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 1or SEQ ID NO:
 3. 12. The method of claim 1, wherein the nucleic acid orexpression vector is administered intramuscularly, subcutaneously,intradermally, and/or into the neoplasm in situ.
 13. The method of claim1, further comprising an administration step of applyingelectroporation.
 14. The method of claim 13, comprising applyingelectroporation to the site of administration of the nucleic acid orexpression vector.
 15. The method of claim 1, further comprisingadministering an immune response potentiator to the mammal.
 16. Themethod of claim 1, wherein the nucleic acid or the expression vector isco-administered with an anti-neoplastic agent or anti-neoplasticregimen.
 17. The method of claim 16, wherein the anti-neoplastic agentis selected from the group consisting of an anti-angiogenic agent, analkylating agent, an antimetabolite, a natural product, a platinumcoordination complex, an anthracenedione, a substituted urea, amethylhydrazine derivative, an adrenocortical suppressant, a hormone, anantagonist, an oncogene inhibitor, a tumor suppressor gene or protein, atherapeutic antibody and an anti-oncogene oligonucleotide.
 18. Themethod of claim 1, wherein the neoplasm to be prevented, treated, ordelayed is selected from the group consisting of adrenal gland, anus,auditory nerve, bile ducts, bladder, bone, brain, breast, centralnervous system, cervix, colon, ear, endometrium, esophagus, eye,eyelids, fallopian tube, gastrointestinal tract, head and neck, heart,kidney, larynx, liver, lung, mandible, mandibular condyle, maxilla,mouth, nasopharinx, nose, oral cavity, ovary, pancreas, parotid gland,penis, pinna, pituitary, prostate gland, rectum, retina, salivaryglands, skin, small intestine, spinal cord, stomach, testes, thyroid,tonsil, urethra, uterus, vagina, vestibulocochlear nerve and vulvaneoplasm, preferably breast cancer, lung cancer, pancreatic cancer,ovarian cancer, gastric cancer, prostate cancer, and melanoma.
 19. Apharmaceutical composition, which comprises: (i) a nucleic acidcomprising a nucleotide sequence encoding a polypeptide, wherein thepolypeptide comprises: (a) a mammalian ErbB3 mutant protein comprisingin comparison to the respective wildtype ErbB3 protein an amino acidsubstitution, which changes the conformation of the extracellular domain(ECD) of said ErbB3 to an extended conformation, (b) a N- and/orC-terminal deletion fragment of (a) comprising at least the ECD of theErbB3 protein; or (c) a variant of (a) or (b), which has at least 80%amino acid sequence identity to the amino acid of (a) or (b) wherein oneor more nucleotide codons encoding (a), (b) or (c) that occur with lowfrequency in proteins expressed in mammalian cells is substituted withnucleotide codons that occur in nucleic acids encoding highly expressedproteins, or which comprises an expression vector comprising saidnucleic acid, and (ii) a pharmaceutically acceptable carrier orexcipient.
 20. The pharmaceutical composition of claim 19, which furthercomprises an anti-neoplastic agent.